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The Isolation and Characterization of the Microbial
Flora in the Alimentary Canal of Gromphadorhina
portentosa Based on rDNA Sequences.
Amy Renee Robertson
East Tennessee State University
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Robertson, Amy Renee, "The Isolation and Characterization of the Microbial Flora in the Alimentary Canal of Gromphadorhina
portentosa Based on rDNA Sequences." (2007). Electronic Theses and Dissertations. Paper 2069. http://dc.etsu.edu/etd/2069
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The Isolation and Characterization of the Microbial Flora in the Alimentary Canal of
Gromphadorhina portentosa Based on rDNA Sequences
______________________
A thesis
presented to
the faculty of the Department of Biological Sciences
East Tennessee State University
In partial fulfillment
of the requirements for the degree
Master of Science in Biology
______________________
by
Amy Renée Robertson
December 2006
______________________
Dr. Karl H. Joplin, Chair
Dr. Bert C. Lampson
Dr. Hugh A. Miller III
Keywords: Microflora, Endosymbionts, Alimentary Canal, Insects, PCR, Cloning, Sequencing,
rDNA Sequences
ABSTRACT
The Isolation and Characterization of the Microbial Flora in the Alimentary Canal of
Gromphadorhina portentosa Based on rDNA Sequences
Amy Robertson
Multicellular organisms are not single individuals but carry a complex natural microflora with
them. This complex’s diversity and function can be considered a distinct ecosystem. Traditional
methods of isolation and identification miss >90% of the actual diversity. This study uses the
gut microflora of the Madagascar hissing roach, Gromphadorhina portentosa, as a model to
examine this ecosystem. Isolated cultured bacteria were used to establish methods for
identifying members of the microflora based on ribosomal RNA sequences. Universal primers
for Eubacterial, Archaeal, and Eukaryotic 16s/18s rRNA were then used for PCR amplification
of total DNA isolated from gut contents. Sequences from isolates were compared using BLAST,
ClustalW, and other programs to recognize the isolates’ identities and place them using a
phylogenetic tree analysis. Eubacterial, Archaeal, and Eukaryotic organisms were found present
in the hissing roach gut which can serve as a model ecosystem since it houses Eubacterial,
Archaeal, and Eukaryotic organisms.
2
CONTENTS
Page
ABSTRACT ....………………………………………………...………………………….
2
LIST OF TABLES ..………………………………………………………………………
5
LIST OF FIGURES ..……………………………………………………………………..
6
Chapter
1. INTRODUCTION …...………………………………………………………
11
2. MATERIALS AND METHODS ...………………………………………….
22
Biochemicals and Bioinformatics Tools …………………………………..
22
Maintenance of Gromphadorhina portentosa …………………………….
24
Methods for Microflora Identification …………………………………….
25
Bacterial Isolation from Roach Tissue and Feces ……………………
25
Single Colony PCR …………………………………………………..
26
Purification of PCR Products ...………………………………………
27
Isolation of Chromosomal DNA from Bacterial Colonies …...……...
28
PCR Reactions Using Chromosomal DNA ………………………….
29
Ligation, Electroporation, and Transformation Reactions …………..
30
Growth of Colonies and Plasmid Extraction ...………………………
32
Insert Confirmation Using Double Digests and PCR ……………….
32
Purification of Samples and Backup PCR …………………………..
33
3
Chapter
Page
Analysis of Sequences …….………………………………………..
33
Extraction of DNA from Roach Gut Content …………………………...
34
Extraction of E. coli DNA as a Control …………………………………
35
Demonstration of DNA Presence …...…...………………………………
36
PCR Amplification .…..…………………………………………………
36
Cloning ………………………………………………………………….
40
Digest Reactions …...………………………………………………
40
Ligation ……………………………………………………………
41
Transformation .……………………………………………………
42
Preparation of Tubes for -80° C Freezer …..………………………
43
Preparation of Stock Plates with Clones ….….........………………
43
Isolation of Plasmid DNA from Clones ......………………………
45
Insert Confirmation …..…...………………………………………
45
Control BamH 1 Enzyme Digest …….……………………………
46
Sequencing ……………………………………………………………
48
3. RESULTS ……………………………………………………………………
49
Identification of Organisms Cultured Directly from Roaches ….....…
49
Culturing of Organisms …..………………………………………
49
Single Colony PCR ...……………….……………………………
51
Chromosomal DNA Isolation …..……………………………………
51
PCR from Combined Bacterial Colony DNA …..……….………
52
Ligation, Transformation, and Plasmid Isolation with PCR Products
53
4
Chapter
Page
Verification of Insert ...…………………………………………
54
Sequencing and BLAST Results ………………………………
55
ClustalW Results ………………………………………………
62
DNA Isolation and Quantification from Roach Gut Tissue ..……….
66
PCR Amplification ………………………………………………….
67
Cloning ...……………………………………………………………
69
Insert Verification …………………………………………………..
70
Control BamH 1 Reaction …………………………………………..
72
Sequencing ………………………………………………………….
73
4. DISCUSSION …………………………………………………………….
74
Research Applications ...…………………………………………...
74
Isolation and Identification of Microflora from Roach Gut and Feces
76
Total DNA Extraction from Roach Tissue ...………………………
78
PCR Amplification .………………………………………………..
79
Cloning and Sequencing …………………………………………..
79
Future Research …………………………………………………..
80
REFERENCES …………………………………………………………………
83
APPENDICES ………………………………………………………………….
86
Appendix A: Abbreviations …………………………………………...
86
Appendix B: Recipes .…………………………………………………
88
Appendix C: Gene Sequences from BLAST ….………………………
91
Appendix D: Phylogenetic Trees from BLAST ..…….……………...
100
5
VITA ...…………………………………………………………………………………
6
106
LIST OF TABLES
Table
Page
1. Reaction Conditions for Tissue and Feces ………………………………………
25
2. Plate Conditions for Tube Samples ……………………………………...............
26
3. Cycles for Single Colony PCR …………………………………………………..
26
4. Dilution of Eubacterial and Eukaryotic Primers ………………………………...
28
5. PCR Reactions for Spooled and Unspooled Chromosomal DNA ………………
30
6. Ligation Reaction Set Up ………………………………………………………..
31
7. Time Constants for Ligation Reactions …………………………........................
31
8. Dilutions for Transformation Plates …………………………………………….
32
9. Initial Sets of Primers …………………………………………………………...
37
10. First Sets of Primers Ordered .…………………………………………………..
37
11. Concentrations and Dilutions of First Sets of Stock Primers .…………………..
37
12. Second Sets of Primers Ordered .………………………………………………..
38
13. Concentrations and Dilutions of Second Sets of Stock Primers ...………………
38
14. Initial Sets of PCR Cycles ………………………………………………………
39
15. Second Sets of PCR Cycles ….………………………………………………….
39
16. Primer Annealing Temperatures ………...………………………………………
39
17. Annealing Temperature Gradient Set Up for PCR …..……………….…………
39
18. PCR Cycles for Gradient ……………….……………………………………….
40
19. Numbers of Digest, Ligation, and Transformation Reactions Performed ..…..…
44
20. Designation of Clones …….…………………………………………..…………
44
7
21. Volumes of Liquid Recovered and Amounts of EtOH and NH4oAc Added …..
45
22. PCR Cycles for Insert Confirmation .…..………………………………………
46
23. Clones Checked for Insert ..………………………………………..…………...
46
24. Clones Sent for Sequencing ..…….……………………..………………………
48
25. BLAST Results for Samples ..…….....…………………………………………
55
26. Values for DNA Isolation Quantifications .……………………….……………
66
27. Results from All PCR Reactions .………………………….…………………..
68
28. Results of Digest, Ligation, Transformation, and Plasmid Isolation Reactions..
70
29. Results of Restriction Digests and PCR Reactions for Insert Verification ..…..
70
30. Results of Clone Sequencing …………………………………………………..
73
8
LIST OF FIGURES
Figure
Page
1. Phylogenetic Tree of Life Determined from rRNA Sequence Comparison……….
12
2. Eubacterial Phylogenetic Tree Based on 16s rRNA Sequence Comparison .…..…
13
3. Laboratory-Raised G. portentosa ............................................................................
24
4. Bacterial Colonies Grown from Roach Feces …….………………………………
49
5. Bacterial Colonies Grown from Roach Gut Tissue ......…….….…………………
50
6. Single Colony PCR Results for Ten Bacterial Colonies .………………………....
51
7. Backup PCR and Chromosomal DNA ……………………………………………
52
8. PCR Results from Bacterial DNA Isolation ..…………………………….……….
53
9. Results of Eukaryotic Digest and PCR ……………………………………………
54
10. Eubacteria Sample #1 Results of BLAST Phylogenetic Tree Analysis ..…………
56
11. Eubacteria Sample #4 Results of BLAST Phylogenetic Tree Analysis ..…………
57
12. Eubacteria Sample #5 Results of BLAST Phylogenetic Tree Analysis …..………
58
13. Eubacteria Sample #7 Results of BLAST Phylogenetic Tree Analysis ……..……
59
14. Eubacteria Sample #8 Results of BLAST Phylogenetic Tree Analysis ……..……
60
15. Eubacteria Sample #10 Results of BLAST Phylogenetic Tree Analysis …………
61
16. Eukaryotic Sample #1 Results of BLAST Phylogenetic Tree Analysis ………..…
61
17. Eubacteria Sample #1 ClustalW Alignment Results ...…….……………...………
62
18. Total Genomic DNA Isolation #4 .…….………………………………………….
66
19. E. coli DNA Extraction Results .…………………………………………………..
67
9
20. PCR Results from Old and New Eubacterial, Archaeal, and Eukaryotic Primers..
68
21. PCR Results for Eubacterial Insert Verification ….………………………………
71
22. PCR Results for Archaeal and Eukaryotic Insert Verification .………....………..
71
23. Digest Results from Control Reaction Checking BamH 1 Activity ………………
73
10
CHAPTER 1
INTRODUCTION
Microbes are an often overlooked part of an ecosystem because of their small size and
lack of morphological characters; however their presence is vital for every kind of known life.
The number of microbial species that exists on the earth today is unknown, and classifying them
becomes a massive project because of their small size, a traditional requirement for colony
isolation, a requirement for a microbiological or biochemical biotype identification, and large
diversity. Molecular techniques have yielded estimates that less than 10% of the organisms in an
ecosystem have been identified using traditional methods (Keeton 2003). In the 19th century the
development of pure culture techniques that allowed scientists to isolate and characterize
prokaryotic organisms were developed by Robert Koch who stipulated that pure cultures of the
pathogenic organism must be isolated from its host and grown in pure culture (Keeton 2003).
With the development of molecular techniques such as PCR and rapid DNA sequencing,
microbial diversity based on DNA sequences greatly simplified the identification of bacteria.
Techniques comparing ribosomal DNA sequences were used to identify a third domain of life,
Archaea, and establish a molecular-sequence based phylogenetic tree that could be used to relate
all organisms (Pace 1997). This information was based on the fact that rDNA sequences are
among the most conserved elements in living things. rDNAs from different organisms were
sequenced using primers specific for each domain, and the alignment of the sequences was
analyzed using a technique known as oligonucleotide cataloging. Oligonucleotide cataloging
takes an RNA molecule and breaks it up into its basic components of (20) oligonucleotides by
slicing at every guanine base using ribonuclease T1 and then analyzing the subfragments.
11
Differences between the sequences were used to relate organisms (Woese 1987). With these
data a molecular-based phylogenetic tree capable of classifying all organisms was created. The
constructed tree demonstrated three primary lines of descent or domains named Eubacteria,
Eukarya, and Archaea. The original rDNA sequences were then able to be rebuilt and were
matched up based on their similarity. As a result, the relationships between all organisms were
described as shown in Figure 1 (Morell 1997, Pace 1997).
EUKARYA
EUBACTERIA
ARCHAEBACTERIA
Figure 1 Universal Phylogenetic Tree Determined from rRNA Sequence Comparison (Adapted
from Woese 1987).
represents organisms that were obtained from PCR and cloning in my
research in relation to this tree.
The compared ribosomal RNA sequences between bacterial domains (Archaea and
Eubacteria) were also used to construct a phylogenetic tree that related known bacteria. The
constructed tree suggested that Eubacteria are monophyletic as shown in Figure 2.
12
EUBACTERIA
Figure 2 Eubacterial Phylogenetic Tree Based on 16s rRNA Sequence Comparison (Adapted
from Woese 1987).
represents organisms that were obtained from PCR and cloning in my
research in relation to this tree.
With the advent of polymerase chain reaction (PCR) techniques, these sequences were
used to investigate the diversity of organisms in environmental or ecological systems (Keeton
2003). However, in every ecosystem examined the diversity of microorganisms had been
grossly underestimated using traditional techniques. PCR techniques indicated that greater than
90% of microorganisms were not being described in these systems (Pace 1997).
All organisms including vertebrates and invertebrates alike harbor a variety of bacteria,
archaeabacteria, and eukaryotes in their digestive tracts. In some organisms, such as cows and
termites, these microflora have been shown to provide necessary functions in the assimilation of
food. Questions still remain concerning the diversity and function of these microflora in the gut.
13
For example, what is the diversity of microbes that inhabit it and what is their function to the
general physiology of the host organism? Could this be considered a contained ecosystem that
can be used to explore ecological questions about the interactions of literally billions of
organisms in complex relations? Insects are good candidates as host organisms because they are
cheap, easy to obtain and rear, and can be easily manipulated in a laboratory.
Roaches are candidate organisms because some species are large, easy to manipulate and
are general foraging omnivores. The gut microflora of two species of cockroach have been
examined (Cruden and Markovetz 1987). Periplaneta americana and Eublaberus posticus have
a diverse diet that includes paper, bread, fruit, fish, putrid sake, cloth, hides, and hair. Over 100
different bacteria have been isolated from or passed through the gut of the cockroach. However,
these microorganisms were isolated from feces or intestine and regarded as normal gut flora.
Although the foregut consists of 50% of the total gut volume, not many microorganisms were
found to colonize the foregut because its pH of 5.4 is unfavorable for microbial activities.
Bacterial isolates from the foregut were not even identified (Cruden and Markovetz 1987).
The midgut, in contrast, showed quite a bit of microbial density. Approximately 108
colonies per midgut were counted when incubated aerobically and 3 x 108 colonies were isolated
when incubated anaerobically. A dilution series isolated on medium containing carboxymethyl
cellulose demonstrated that the midgut of both roach species contained 106 bacteria that grew
under aerobic conditions and 107 that grew anaerobically. The most common microbes that were
isolated from the midgut included anaerobes, Enterobacter agglomerans and Klebsiella oxytoca,
and Citrobacter freundii. Electron microscopic examination of the midgut also showed that
several types of insects harbor bacteria between the peritrophic membrane and midgut epithelium
with occasional protozoa and spirochetes also present (Cruden and Markovetz 1987).
14
The hindgut in contrast, only harbors anaerobic bacteria. The bacteria consistently
isolated included organisms Clostridium sporogenes, Fusobacterium varium, Eubacterium
moniliforme, Peptococcus variabilis, Peptostreptococcus productus, and Bacteriodes sp.
Bacteria frequently isolated from the cockroach hindgut include Acidaminococcus fermentans,
Propionibacterium avidum, Bifidobacterium sp., Clostridium bifermentans, Lactobacillus sp.,
Butyrivibrio sp., Coprococcus sp., and Ruminococcus sp. Even though these bacteria can be
identified by species, many of the isolates cannot, especially when nonstandard enrichments are
used such as stimulation with an inorganic phosphate. In addition to bacterial species in the
hindgut, various types of ciliated protozoans were present as well as methanogens that live
within the protozoans. The protozoans comprised approximately 0.2% of the population in the
hindgut lumen. Methanogens included organisms with an ultrastructure similar to the genus
Methanospirillium (Cruden and Markovetz 1987).
In addition to bacterial identification in the cockroach gut, ecological studies were done
on the methanogens and ciliated protozoans. These studies investigated how methanogens that
resemble organisms from the genus Methanobacter that live in the ciliated protozoan
Nyctotherus ovalis in the gut of the American cockroach Periplaneta americana form a
mutualistic relationship between the two organisms (Gijzen et al. 1991). The presence of the
protozoan in the alimentary canal of the cockroach represents a production of methane. When
metronidazole that inhibits methanogenesis was added to the cockroach drinking water
significant changes were observed. Metronidazole was added for a period of 3 months and
caused complete inhibition of methane production. Metronidazole was also added for 8 days
and the N. ovalis cells were monitored every day and the protozoan was eliminated from the
roach hindgut after 3 days. These studies show that the roach gut or alimentary canal can serve
15
as an isolated ecosystem and that the elimination of even one or two of the organisms can alter
the system (Gijzen et al. 1991).
This system can also be used to investigate how the organisms interact with the host
(ecosystem) (Gijzen et al. 1991). The elimination of N. ovalis from the cockroach hindgut
demonstrated that when the cockroaches were raised without N. ovalis, they showed reduced
body weight, increased generation time, and absence of methane production (Gijzen and
Barugahare 1992). When the protozoan-free roaches were fed a hindgut suspension that
contained N. ovalis as well as methanogens, methane production became normal and insect
weight was reestablished during the second generation of insects. When protozoan-free roaches
were fed a hindgut suspension with protozoans but no N. ovalis methane production grew to only
20% of the normal level. Roaches fed a suspension of bromoethanesulfonic acid reduced
methane production to 2% of the normal level. This caused a shift in the hindgut fermentation
pattern as well as an increase in propionate production. This shows that the ciliated protozoan N.
ovalis plays a major role in methane production in the cockroach gut as well as in metabolism.
This specific organism is of ecological importance because it contributes greatly to the overall
metabolism and nutrition of the cockroach. Roaches are thought to be a major contributor of
much of the world’s methane. The interaction of these endosymbionts with each other and their
host may point to new ways of how to control insects and demonstrate their importance to the
ecosystem (Gijzen and Barugahare 1992).
Many of these studies were done in the 1980s and early 1990s before advanced molecular
techniques were known. These early studies were based on Koch’s principle of pure
cultures/isolation and were limited because only a handful of microorganisms can be cultured
using traditional methods (Pace 1997, Keeton 2003). Molecular techniques have allowed
16
biologists to greatly expand the classification and identification of organisms without these
limitations (Pace 1997).
The early studies of environmentally isolated microbes were limited to pure cultures that
can be grown in the laboratory; however, most of the isolates could not be identified and
characterized through this system. Experiments on different environments have shown that more
than 99% of microorganisms cannot be cultured by traditional techniques. With the development
of rRNA sequencing to classify microbes, only the rRNA sequence is required to identify the
prokaryote and be placed on the phylogenetic tree (Pace 1997). Ribosomal genes can be used
because of their highly conserved function. Ribosomal sequences obtained by cloning DNA that
has been isolated directly from the organism must be sorted from other isolates through a cloning
procedure that allows each one to be sequenced. Ribosomal RNA’s highly conserved nature
allows for the use of “universal” PCR primers capable of annealing to conserved rRNA
sequences from the three phylogenetic domains. The sequences can be compared with known
sequences, analyzed, and placed on the phylogenetic tree based on the similarities in the
sequences (Pace 1997).
A study that estimated bacterial biodiversity was done using rDNA PCR primers
(Marchesi et.al. 1998). In the past, PCR primers often failed to work with some of the samples
such as deep sea sediments, oral bacteria, and epilithon (biofilms that are associated with stones
in moving water environments). Other primers were designed that were universal for Domain
Eubacteria based on previous research on bacterial evolution. As a result of the primer redesign,
a new array of bacterial species was identified. These included organisms from the genera
Micrococcus and Eubacterium in addition to δ-proteobacteria and bacteria from moving water
17
samples and deep sea sediments. This study was the first that used universal Eubacterial primers
for PCR amplification that amplified a larger number of bacterial genes (Marchesi et.al. 1998)
With the ability to identify increasing diverse prokaryotic species, attention was turned to
eukaryotic species. The biodiversity of eukaryotic phytoplankton in the oceans revealed a vast
array of eukaryotic organisms through the use of 16S small subunit rRNA amplification (Moriera
and López-García 2002). It was previously shown within the Eubacteria domain that 13
divisions have been cataloged. There are, however, many organisms that have not been cultured
within the two major Archaeal kingdoms Chrenarchaeota and Euryarchaeota. Several plastid
genes were amplified which allowed the identification of a large number of photosynthetic
lineages related to the classes Bacillaryophyceae, Cryptophyceae, Prymnesiophyceae,
Chrysophyceae, and Prasinophyceae (Moreira and López-García 2002). This study also
uncovered many new lineages as well. Some of these were affiliated to non-photosynthetic
groups, one Pseudo-nitzschia like diatom, dinoflagellate phylotypes, and lineages that are not
closely related to any known organisms. The most striking discovery was the assemblage of
very diverse sequences that comprised the majority of their clones. These sequences formed two
distinct clusters within the alveolates. This study demonstrates that the ocean is a vast ecosystem
that contains microscopic eukaryotic organisms in greater diversity than previously thought
(Moreira and López-García 2002).
Studies done to determine the biodiversity of microbes in the oceans have assessed
marine microorganism diversity though sequencing ribosomal genes through the use of PCR
amplification and have identified 20 major phyla in Bacteria and Archaea plus thousands of new
taxonomic units. Larger microbes that were sequenced and divided into 5000 autotrophic and
1500 species based on morphology or outward characteristics (Falkowski and de Vargas 2004).
18
Whole genome sequencing is another technique that has been applied to environmental-pooled
DNA samples (Venter et al. 2004). Picoplankton, or marine eukaryotes are thought to be the
most abundant eukaryotes on earth, thus they would have a very wide range of genetic diversity.
Total DNA sequencing and computational genomics has helped to identify more than 1.2 million
new genes from organisms isolated from the Sargasso Sea. (Díez, Pedrós-Alió and Massana
2001, Venter 2004). Recently, approximately 1.6 million DNA sequences have been obtained
from the Sargasso Sea and analyzed (Venter et al. 2001). The analysis first focused on the wellsequenced genomes by characterizing scaffolds, or proteins that remain when chromosomes are
digested away (Lawrence 2001). The results were 333 scaffolds that comprised 2226 contigs
(DNA sequences that are assembled from overlapping shorter sequences to form a continuous
one) (Lackey 2001) that spanned 30.9 megabase (mbp) pairs. This accounted for roughly
410,000 reads, or 25% of the assembled data set. However, PCR studies have been discovered to
be somewhat biased because not all genes will amplify with the same universal primers. The
researchers, using the shotgun sequencing method, identified 1164 16s rRNA genes (Venter et al.
2004). Currently, there have been 36 to 38 phylogenetic divisions of microbes discovered based
on 15,000 rRNA sequences from both cultured and environmental organisms. However, only
13 of these divisions have been encountered in environmental surveys and have not been able to
be cultured using traditional methods. Some of these sequence-defined divisions have <10
sequences to represent them so the diversity of these organisms is vastly unknown (Dojka et al.
2000).
These studies still leave a lot of questions unanswered. What is the total diversity of
bacteria, archaeabacteria, and eukaryotes in the environment? Could a more comprehensive
description of species in the insect alimentary canal be identified through the use of rDNA
19
sequencing and PCR instead of traditional culturing methods? Could the insect gut serve as a
model ecosystem?
My project is to describe the components of the alimentary canal of insects by examining
the biodiversity of microbes present, identify them based on sequences from the extraction of
rDNA, and develop this organismal complexity as an isolated ecosystem. The above studies
provide evidence that it is possible to obtain many ribosomal genes from organisms comprising
the three phylogenetic domains. The PCR product in this experiment should yield three different
size products using specific primers for the Eukarytic, Eubacteria, and Archaeal domains.
I examined the types of microflora in the alimentary canal of the Madagascar hissing
roach, Gromphadorhina portentosa, through the use of PCR, cloning, and sequencing. The gut
microflora of insects has not been investigated significantly using these molecular techniques
and this study will attempt to answer the question of what kinds of microflora can be found from
the gut of an insect. Also, could an insect alimentary canal serve as a model for an ecosystem?
This ecosystem would comprise organisms from all three phylogenetic domains and would
explore the network of symbiotic relationships between organisms.
My working hypothesis states that when the biodiversity of microbes present in the
alimentary canal of Gromphadorhina portentosa is examined, organisms comprising all three
phylogenetic domains are present. These organisms can then be compared with other sequences
from other organisms and placed on the phylogenetic tree produced by Woese that contains all
three domains of life (Eubacteria, Archaeabacteria, and Eukarya). A prediction is that there will
be some species of microflora that have not been identified, or if they have been identified, little
is known about them. I identified bacteria that were isolated using traditional methods and then
used total DNA from the roach’s gut for PCR with universal primers, cloning, and DNA
20
sequencing. Sequences obtained were analyzed using BLAST, ClustalW, and other programs
and placed on the phylogenetic tree comprising all three phylogenetic domains based on their
similarities with other sequences.
21
CHAPTER 2
MATERIALS AND METHODS
Biochemicals and Bioinformatics Tools
For roach gut tissue extraction, insect saline (Appendix B) was used to clean roach tissue
and the gut was extracted with dissecting scissors. DNA was isolated from the gut tissue using
DNAzol (Molecular Research Center Inc.,Cincinnati, OH) chloroform (Fisher Scientific,
Fairlawn, NJ), phenol (Sigma Aldrich, St. Louis, MO), lysozyme (100 µg/µl) (Sigma-Aldrich,
St. Louis, MO), 10% SDS, and Proteinase K (100 µg/µl). Isolated DNA was stored in TE buffer
(recipe in Appendix B).
ReadyMixTM Taq PCR Reaction Mix with MgCl2 (Sigma-Aldrich, St.Louis, MO) and
DIUF water, primers designed from Operon Biotechnologies Inc. (Huntsville, AL) and Sigma
Genosys Custom Oligos (Sigma-Aldrich Co., The Woodlands, TX), and extracted template DNA
were used for PCR. PCR products were extracted from the 1% gel using GenElute Agarose Spin
Columns (Sigma-Aldrich, St. Louis, MO) and purified using 7.5 M NH4oAc and 95% EtOH.
Blue dye (Finnzymes, Espoo, Finland) was used to determine the DNA’s location on the gel.
Molecular weight markers (DNA ladders) included λ/HindIII, ExACTGene Low Range Plus
DNA Ladder (Bioline USA Inc., Randolph, MA), and λDNA-HindIII/øx-HaeIII (Finnzymes,
Espoo, Finland)).
Cloning used 38i and 28i (5 µg/ml) LITMUS cloning vectors (New England Biolabs,
Beverly, MA), purified PCR product, BamH 1 enzyme (2500 units) (Fisher Scientific, Pittsburg,
PA), and Sal 1 enzyme (2000 units) (Fisher Scientific, Pittsburg, PA). QIAGEN® PCR Cloning
22
Kit (40 rxns) (Qiagen Inc., Valencia, CA) was used and included pDrive Cloning Vector (2.0 µg,
50 ng/µl).
Ligation reactions used Quick Stick Ligation Kit (50 reactions) (Bioline USA Inc.,
Randolph, MA) containing T4 DNA ligase (50 μl). Another kit (Promega Corporation, Madison,
WI) contained pGEM®-T Easy Vector (1.2 µg).
Transformation reactions were done using One Shot® Mach1TM-T1R Chemically
Competent E. coli cells (Invitrogen Life Technologies, Carlsbad, CA), SOC medium (Invitrogen
Life Technologies, Carlsbad, CA), and X-gal (20 µg/µl) (Fisher Biotech, Fair Lawn, NJ).
Plasmid DNA isolations used QIAprep® Spin Miniprep Kits (50 rxns) (Qiagen Inc., Valencia,
CA) containing spin columns (50),
LB broth (Fisher Scientific Inc., Fair Lawn, NJ) was used with ampicillin (25 µg/µl), LB
agar (Fisher Scientific Inc., Fair Lawn, NJ). Ampicillin (Fisher Scientific, Fair Lawn, NJ) and
Sigma-Aldrich (St. Louis, MO)) and X-gal plates were prepared according to the recipes in
Appendix B.
Gels were run with 1X TBE buffer (prepared according to recipe in Appendix B) and 1%
agarose (Fisher Biotech Inc., Fairlawn, NJ). These gels were stained in 10 µg/µl EtBr (Fisher
Scientific, Fair Lawn, NJ), (EtBr was also prepared according to the recipe in Appendix B). The
gels were photographed using the program LabWorks 4.0. PCR products were further purified
for sequencing using GENECLEAN® Turbo Kit (100 preps) (Qbiogene Inc., Solon, OH).
For DNA sequencing, NCBI’s BLAST website was used to identify organisms and
construct phylogenetic trees, ClustalW (EMBL-EBI) was used to align the sequences, and the
University of Tennessee, Knoxville’s services were used to sequence the DNA.
23
Maintenance of Gromphadorhina portentosa
Madagascar hissing roaches, Gromphadorhina portentosa, are large wingless insects
native to the island of Madagascar off the eastern coast of Africa. They live on the forest floor as
omnivorous scavengers. One of the distinguishing characteristics of this insect is its ability to
produce sound through spiracles in its abdomen. This occurs when the roach is disturbed by a
predator or when males come in contact with each other (Clark and Shanklin 1995).
G. portentosa colonies originated from Ohio State University’s entomology department
and were maintained since 1994 at East Tennessee State University. Roaches were fed dry dog
food and tap water ad libitum in a plastic container. Colonies were kept at 27° C in constant
darkness. G. portentosa is shown in Figure 3.
Figure 3 Laboratory-Raised G. portentosa
24
Methods for Microflora Identification
Bacterial Isolation from Roach Tissue and Feces
Microorganism isolation was from feces and gut tissue collected from immature roaches.
Once extracted, the feces were placed into a test tube with 1 ml BHI broth and mixed to remove
organisms. The broth was streaked onto four plates—two blood agar and two nutrient agar. One
blood agar and one nutrient agar plate were placed into an anaerobic chamber with a gas pack.
Ten ml of water was added to this gas pack (containing NaBH4) causing O2 to be removed. The
other two plates were placed into a jar with a lit candle that resulted in elevated CO2 levels.
The gut and contents were similarly extracted and placed into a sterile tissue
homogenizer with 2 ml BHI broth and ground. The extracted samples were streaked onto blood
agar and nutrient agar plates and placed in an aerobic jar with a candle to raise CO2 levels. Both
the candle jar and the anaerobic chamber were incubated at 30° C. The culture conditions for the
samples are shown in Table 1.
Table 1 Reaction Conditions for Tissue and Feces
Anaerobic chamber
Anaerobic chamber
Candle Jar
Candle Jar
Blood Agar
Feces
Roach #1 Tissue
Feces
Roach #2 Tissue
Nutrient Agar
Feces
Roach #1 Tissue
Feces
Roach #2 Tissue
The ground up tissue and feces samples were individually mixed with BHI (800 µl of
each sample) and placed into glass vials with 200 µl 75% glycerol (to prevent freezing at very
low temperatures) (18.75% glycerol final concentration). Samples were stored at -80° C.
25
Single Colony PCR
Single colony PCR was performed on 10 of the individual colonies that grew (10
different ones). For this reaction, ten 1.5 ml microcentrifuge tubes were labeled 1-10 and 10 µl
PCR water was added to each of them. In each of these tubes a single different bacterial colony
was added using an inoculation needle and was mixed thoroughly in the water. The plate
conditions for these tube samples are displayed in Table 2.
Table 2 Plate Conditions for Tube Samples
Tube #1
Tube #2
Tube #3
Tube #4
Tube #5
Tube #6
Tube #7
Tube #8
Tube #9
Tube #10
Feces/Anaerobic Chamber/Blood Agar
Feces/Anaerobic Chamber/Blood Agar
Feces/Anaerobic Chamber/Blood Agar
Roach #2 Tissue/Candle Jar with Elevated CO2/Nutrient Agar
Roach #2 Tissue/Candle Jar with Elevated CO2/Blood Agar
Feces/Anaerobic Chamber/Blood Agar
Roach #2 Tissue/Candle Jar with Elevated CO2/Blood Agar
Roach #1 Tissue/Anaerobic Chamber/Blood Agar
Roach #1 Tissue/Anaerobic Chamber/Blood Agar
Roach #1 Tissue/Anaerobic Chamber/Blood Agar
For the PCR reaction, 1 µl of liquid from each sample was placed into a clean 0.2 ml
PCR tube and mixed with 1 µl each of FB and RS Eubacterial primers (BamH1 and Sal1
restriction sites), 12.5 ReadyMixTM Taq PCR Reaction mix, and 9.5 µl PCR water. These
reactions were mixed by vortexing and placed into a thermocycler. The PCR cycles are
displayed in Table 3.
Table 3 Cycles for Single Clony PCR
95°C/2 min
95°C/1 min
4°C/park
50°C/2 min
72°C/2 min
1 cycle
30 cycles
26
When the PCR cycles were complete, 10 µl of each PCR product sample was run in two
1% agarose gels at 104 volts for 45 minutes. Gels were stained in 200 ml 1X TBE buffer with
10 µl EtBr (10 µg/µl) for 30 minutes. They were viewed on a transilluminator and photographed
using LabWorks 4.0. The PCR products (bands) were cut out from the gel and purified by
adding DIUF water (90 µl) to each sample and mixing with 250 µl ice cold 95% EtOH to
precipitate the DNA. The samples were placed at -20° C overnight and spun for 15 minutes at
14,000 rpm. The pellet was allowed to air dry briefly, and the DNA redissolved in 15 µl DIUF
water. A repeat PCR reaction was performed using 1 µl DNA from the single colony dissolve (1
bacterial colony + 10 µl DIUF water), 12.5 µl ReadyMixTM Taq PCR Reaction mix, 2 µl FB and
RS primers (Eubacterial with BamH 1 and Sal 1 sites), and 9.5 µl DIUF water. They were run in
the thermocycler with the same cycles as Table 3, run on a gel, and purified using the PCR
purification protocol.
Purification of PCR Products
Further purification of these products was performed using the GENECLEAN® Turbo
Kit according to the kit instructions. For these reactions, 5 volumes of Turbo Salt Solution (75
µl) was added to each of 10 tubes. Each of these reactions was transferred to a separate Turbo
cartridge in a 2 ml catch tube. The tubes were centrifuged for 5 seconds at 13,200 rpm. After
centrifugation 500 µl prepared TurboWash was added to each tube and centrifuged for 13,000
rpm for 5 seconds. The flowthrough was discarded and the tubes centrifuged for an additional 4
minutes (13,200 rpm) to force the remaining wash through the tube. After this remaining wash
was discarded, the filters were placed into new, clean Turbo catch tubes and 50 µl Turbo Elution
Solution was added to the center of each tube and allowed to sit for 5 minutes at room
27
temperature. The tubes were centrifuged for 1 minute at 13,200 rpm and eluted was DNA stored
in the freezer at -20° C. The DNA was further purified for sequencing using the 95% EtOH
precipitation method used previously. These PCR products were run in 5µl aliquots on a 1%
agarose gel to demonstrate that DNA was present. Samples with DNA present were sent for
sequencing (UT Knoxville sequencing lab). Eubacterial and Eukaryotic primers (Table 10) (with
BamH 1 and Sal 1 sites) were diluted to 5 µM concentration according to Table 4 in order to be
used for sequencing from single colony PCR.
Table 4 Dilution of Eubacterial and Eukaryotic Primers
Primer
Eub 27FB
Eub 1492RS
Euk FB
Euk RS
Amount Stock Added
5 µl
5 µl
5 µl
5 µl
Amount DIUF H20 Added
28.6 µl
32.9 µl
36.2 µl
31.9 µl
Total Volume
33.9 µl
37.9 µl
41.2 µl
36.9 µl
Isolation of Chromosomal DNA from Bacterial Colonies
Two plates were obtained, one plate from the anaerobic jar and one from the candle jar
the bacterial colonies scraped off of them, and the bacteria washed in 500 µl mM Tris (pH 8.0).
After washing, the liquid was poured off and the bacteria were stored at -20° C. The colonies
were resuspended in two tubes each with 160 µl 25% sucrose solution in 50 mM Tris in 1.5 ml
microcentrifuge tubes before they were finished thawing. Lysozyme (60 µl, 10 mg/ml) was
added to each tube, shaken to mix, and placed in a hot water bath at 37° C for 30 minutes. After
incubation, 67 µl 0.25 M EDTA was added to each tube as well as 50 µl 10% SDS and 50 µl
Proteinase K (1 mg/ml) to break up cell membranes . The tubes were inverted gently until the
solution became viscous and clear. They were incubated in the hot water bath at 37° C for 30
minutes. After incubation, 150 µl 50 mM Tris (pH 8.0) to stabilize pH was added and the tubes
28
mixed gently. Phenol (500 µl) was then added remove and denature proteins from the DNA, the
tubes mixed vigorously, and placed on ice 1-2 minutes. They were centrifuged 5 minutes at
15,000 rpm at 4° C. The aqueous (top) layer from each tube was saved and re-extracted with
phenol (450 µl). The tubes were mixed vigorously, incubated on ice 1-2 minutes, spun at 4° C at
15,000 rpm for 5 minutes, and the aqueous layer saved. Chloroform (450 µl) was added to each
of the tubes to remove the phenol. They were shaken vigorously, incubated on ice 1-2 minutes,
and spun at 4° C at 15,000 rpm for 2.5 minutes. The aqueous layer was saved and re-extracted.
Chloroform was treated as before. The aqueous layer from each tube was saved and 1/10
volume 10 M NH4oAc was added to convert the DNA to a salt instead of an acid. Ice cold 100%
EtOH was added to precipitate the DNA salt. The tubes were mixed well and the DNA was
spooled out with a 0.1-10 µl plastic pipet tip and placed into 1.5 ml tubes with 400 µl 70%
EtOH. These tubes were spun at 4° C at 15,000 rpm for 30 seconds. The EtOH was aspirated
off using a Pasteur pipet and the DNA briefly allowed to air dry. It was resuspended in 100 µl
0.1X TE buffer (pH 8.0) to dissolve the DNA. The tubes were stored at 4° C overnight. The
chromosomal DNA was diluted 1/5000. Leftover unspooled DNA from the tube was spun in the
centrifuge for 15 minutes at 10,000 rpm, the EtOH aspirated off using a Pasteur pipet, briefly
allowed to air dry, and resuspended in 50 µl DIUF water.
PCR Reactions Using Chromosomal DNA
PCR reactions were performed using the diluted spooled chromosomal DNA and the
leftover unspoiled DNA from the extraction. For this reaction, primers (with BamH 1 and Sal 1
sites, Table 10) representing the three phylogenetic domains (Eubacteria, Archaea, Eukarya)
were used. Six 0.5 ml microcentrifuge tubes were obtained and labeled accordingly. These
reactions were mixed up and run with the same PCR cycles as Table 5 demonstrates.
29
Table 5 PCR Reactions for Spooled and Unspooled Chromosomal DNA
Amount DNA
Eub Spooled
Chromosomal
DNA
Eub Unspooled
Chromsomal
DNA
Arch Spooled
Chromosomal
DNA
Arch Unspooled
Chromsomal
DNA
Euk Spooled
Chromosomal
DNA
Euk Unspooled
Chromsomal
DNA
5 µl
Amount
Taq Mix
12.5 µl
Amount
Primers
1 µl FB
1 µl RS
Amount
Water Used
5.5 µl
Total
Volume
25 µl
3 µl
12.5 µl
1 µl FB
1 µl RS
7.5 µl
25 µl
5 µl
12.5 µl
1 µl FB
1 µl RS
5.5 µl
25 µl
5 µl
12.5 µl
1 µl FB
1 µl RS
5.5 µl
25 µl
5 µl
12.5 µl
1 µl FB
1 µl RS
5.5 µl
25 µl
5 µl
12.5 µl
1 µl FB
1 µl RS
5.5 µl
25 µl
After the PCR cycles were complete, the six samples were run on a 1% agarose gel at 104
volts for 45 minutes. The gel was stained in 200 ml TBE + 20 µl EtBr (10 µg/µl) for 30 minutes.
The PCR products were purified using the same process as above.
Ligation, Electroporation, and Transformation Reactions
For ligation, five different reactions were performed with the samples shown in Table 6.
The reactions were vortexed and placed in the refrigerator at 4º C overnight.
30
Table 6 Ligation Reaction Set Up
Eub Sp. Chr.
Euk Sp. Chr.
Eub Unsp. Chr.
+ Control
- Control
2x Rapid
Ligation Buffer
5 µl
5 µl
5 µl
5 µl
5 µl
pGEM® -T
Vector
1 µl
1 µl
1 µl
1 µl
1 µl
PCR DNA
from Tubes
3 µl
3 µl
3 µl
---
T4 DNA
Ligase
1 µl
1 µl
1 µl
1 µl
1 µl
dH2O
Added
---1 µl
3 µl
Four vials (100 µl each) of DH5α electrocompetent E. coli cells were thawed on ice. One
of the vials was divided into 50 µl cells each for the controls. After thawing, 1.5 µl of ligation
reaction was added to each vial and each reaction was transferred to 2 mm electroporation
cuvets. Electroporation was performed at 2500 volts. The time constant for each of the different
reactions was recorded as shown in Table 7.
Table 7 Time Constants for Ligation Reactions
Reaction
Time
Constant
Eub Sp. Chr.
4.8 s
Euk Sp. Chr.
4.4 s
Eub Unsp. Chr.
4.8 s
+ Control
5.2 s
- Control
5.2 s
Five 16 ml test tubes were obtained and 500 µl SOC medium was added to the cuvets,
mixed, and then extracted using a Pasteur pipette. The mixture from each cuvet was then placed
into the test tubes and allowed to incubate at 37º C for 1 hour. Eleven LB + amp plates were
prepared using 75 µl IPTG and 25 µl X-gal on 9 of them. Two plates containing carbinicillin
were also used. The IPTG was smeared on the plates and allowed to dry, and then the X-gal was
added. After an hour the transformation reactions were diluted and inoculated onto the plates
according to Table 8. The transformations were then placed in the incubator at 37° C overnight.
31
Table 8 Dilutions for Transformation Plates
Eub Sp. Chr.
Euk Sp. Chr.
Eub Unsp. Chr.
+ Control
- Control
Vol/Plate
100/50 µl
50 µl
50 µl
50 µl
50 µl
Type Plate
LB + Amp 50
LB + Amp 50
LB + Amp 50
LB + Amp 50
LB + Amp 50
X-gal + IPTG
Yes
Yes
Yes
No
No
# 100 (Undiluted)
1
1
1
1
1
# 102
2
2
2
0
0
Growth of Colonies and Plasmid Extraction
Three of the plates (Eub Sp. Chr., Euk Sp. Chr., and Eub Unsp. Chr.) were placed in the
refrigerator overnight. Two and a half ml LB + 2.5 µl ampicillin (25 µg/µl) was added to each of
thirty-two 18-ml test tubes. Each of 32 individual colonies was taken from these plates using an
inoculation needle dipped in LB broth to ensure the bacteria would come off the plates more
easily. Nine colonies came from the Eubacterial Unsp. Chr. plate, 4 colonies came from the
Eukaryotic Sp. Chr., and 17 colonies came from the Eubacterial Sp. Chr. plate. The other two
colonies were blue ones (controls that did not contain the 16s/18s ribosomal gene insert) that
came from the Eubacterial Unsp. Chr. plate (100 dilution) and Eukaryotic Sp. Chr. plate (100
dilution). These tubes were placed in the shaking incubator at 200 rpm at 37° C overnight.
Plasmids were isolated from the colonies that grew with QIAGEN QIAPrep® spin miniprep kits.
At the end of the protocol, instead of allowing the samples to stand for 1 minute before
centrifugation (elution step), they were allowed to stand 15 minutes.
Insert Confirmation Using Double Digests and PCR
A double digest reaction using BamH 1 and Sal 1 restriction enzymes was performed on
the Eubacterial (Unsp. Chr.) and Eukaryotic (Sp. Chr.) samples. For these reactions, 7 µl DNA,
32
1 µl BamH 1 enzyme, 1 µl Sal 1 enzyme, 4 µl Buffer E, and 9 µl PCR + DIUF H2O were mixed
by pipetting in labeled 0.5 ml microcentrifuge tubes. The tubes were placed at 37° C overnight.
The samples were run on two 1% agarose gels as above. PCR reactions were also performed
with these 9 samples as well as the controls. The Eukaryotic samples were placed in the
thermocycler and the cycles from Table 5 were used. For the Eubacterial samples, the cycles
shown in Table 3 were used. These samples were run on 1% agarose gels as above. They were
stained in 300 ml TBE with 20 µl EtBr (10 µg/µl) for 20 minutes. These samples were also
purified using 2.5 volume ice cold 95% EtOH and 0.5 and 0.1 volumes NH4oAc. Once these
two reagents were added to the samples, they were stored in the freezer at -20° C. They were
then centrifuged at 10,000 rpm for 15 minutes. The EtOH was aspirated off using a Pasteur pipet
and 50 µl DIUF H2O was added.
Purification of Samples and Backup PCR
The three PCR samples were purified as done previously using GENECLEAN® Turbo
Kit, EtOH, and NH4oAc. A repeat PCR reaction was also performed using the leftover unspooled
chromosomal DNA from the extraction as well as a 1/5000 dilution of the spooled chromosomal
DNA. A new PCR reaction was performed and a gel run with these products. The Eubacterial
band was cut out and gel purified. Another PCR reaction was then run and the Eukaryotic band
extracted (gel purified).
Analysis of Sequences
Sequences were copied from the BioEdit program (Ibis Therapeutics) into a Microsoft
Word document. The FB sequences were taken for all 13 samples (Eub and Euk) and the first
33
500-900 base pairs were recopied. The unnecessary N’s from the sequences were also deleted.
The sequences were then placed into the BLAST website (NCBI), their relationships were
observed, and phylogenetic trees were constructed from the relationships with other organisms.
The edited sequences from these organisms were compared with the sample sequences in
ClustalW (EMBL-EBI).
Extraction of DNA from Roach Gut Content
For DNA isolation, a large adult male G. portentosa was obtained and anesthetized in the
freezer. The gut content was dissected in insect saline and weighed in a small, plastic tube. Four
ml DNAzol was added and then ground with mortar and pestle. Four hundred μl 10% SDS were
then added and ground more. This mixture was incubated at 65° C for 30 minutes. After
incubation, 1.35 ml chloroform was added and centrifuged 20 minutes at 10,000 rpm.
Approximately 4 ml of a brown liquid was collected and 4 ml chloroform and 4 ml phenol were
added. The mixture was shaken and centrifuged for 20 minutes at 10,000 rpm. After
centrifugation the mixture was placed in the refrigerator at 4º C. The mixture was measured and
equal volumes of phenol and chloroform were added. The mixture was shaken and centrifuged
for 20 minutes at 10,000 rpm. The brown supernatant was taken off and measured and equal
volumes of phenol and chloroform were added again and centrifuged for 20 minutes at 10,000
rpm. The brown supernatant was collected and an equal volume of chilled isopropanol was
added and stored in the refrigerator overnight. The mixture was centrifuged 20 minutes at
10,000 rpm and the pellet was redissolved in 1 ml TE buffer. The solution was aliquoted into
two microfuge tubes. The DNA was precipitated by the addition of 60 μl NH4oAc (7.5 M) and
1200 μl 95% EtOH. The tubes were refrigerated and then centrifuged for 20 minutes at 10,000
34
rpm in the tabletop centrifuge. The supernatant was discarded and 100 μl TE buffer was added
to each tube to dissolve the DNA. The DNA solution was again extracted with 50 μl chloroform
and 50 μl phenol and centrifuged 20 minutes at 10,000 rpm. The aqueous layer was
reprecipitated with one-tenth volume of 7.5 M NH4oAc and 2 volumes of 95% ethanol. After
refrigeration, the DNA was pelleted by centrifugation 20 minutes at 10,000 rpm and the
supernatant poured off. The pellet was dissolved in 200 μl TE buffer and further purified by the
addition of 250 µl DNAzol and 75 µl chloroform. After centrifugation for 20 minutes at 14,000
rpm, the top layer (aqueous) was recovered. An equal volume of isopropanol (~500 µl) was
added and the mixture was placed in the refrigerator overnight. It was centrifuged for 20
minutes at room temperature at 14,000 rpm and the pellet was resuspended in 50 µl TE buffer.
Aliquots of 5 µl, 10 µl, and 20 µl were run on a 1% agarose gel.
Extraction of E. coli DNA as a Control
Escherichia coli DNA was isolated as a control for use with Eubacterial primers. A
solution (10 ml) of LB broth was incubated overnight at 37°C with E. coli bacteria. The solution
was spun to pellet the bacterial cells. After the cells were pelleted a 100 µl lysozyme solution
was added and placed in a shaker at room temperature for 15 minutes. A volume of 800 µl of
10% SDS was added and the mixture incubated at 65°C for 20 minutes. A volume of 2 ml
DNAzol was added followed by 750 µl chloroform. The mixture was centrifuged at 10,000 rpm
for 20 minutes and the supernatant recovered (3 ml). An equal volume of isopropanol was
added, placed in the refrigerator for 1 hour, and centrifuged for 20 minutes at 10,000 rpm. The
supernatant was poured off and 100 µl TE buffer was added to dissolve the DNA. A 1% agarose
gel was run with an aliquot of 10 µl E. coli DNA, 2 µl EtBr (10 µg/µl), and 2 µl blue dye.
35
Demonstration of DNA Presence
The presence of DNA was demonstrated by running samples on a 1% agarose gel (0.30
gram agarose and 30 ml 1X TBE buffer). The agarose was melted in buffer by boiling for 2
minutes and poured into a gel mold. After the gel had hardened, the DNA samples were loaded
into the gel along with a molecular marker (λ cut with Hind III) with 2 µl EtBr (10 µg/µl) and 2
µl blue dye. After the dye was ¾ of the way down the gel it was stained in 200 ml TBE with 10
µl EtBr (10 µg/µl) for 15 minutes in the shaker incubator. It was viewed under the
transilluminator and photographed using LabWorks 4.0.
DNA was quantified using a spectrophotometer and 0.5 ml glass cuvets. The first cuvet
had 500 μl dH2O and served as a blank. The second cuvet contained 5 μl DNA and 495 μl dH2O.
This DNA could then be used for PCR analysis.
PCR Amplification
PCR amplification was performed using several sets of primers representing the three
phylogenetic domains (Eubacteria, Archaea, Eukarya). Initial sets of primers were used (Joplin
1998) as well as second and third sets (DeLong 1992). The initial sets of primers from Table 9
and the first ordered sets from Table 10 contained BamH 1 and Sal 1 restriction sites. The
second ordered sets, however, contained only BamH 1 sites (Table 12). Primers were diluted
accordingly (Tables 11 and 13) to obtain a 10 mM concentration.
36
Table 9 Initial Sets of Primers (Joplin, 1998)
Specific Domain
Eubacteria 1a
Eubacteria 1b
Archaebacteria 1a
Archaebacteria 1b
Eukarya 1a
Eukarya 1b
Primer Name
63FB
1357RS
21FB
958RS
Euk FB
Euk RS
Primer Sequence (5’ to 3’)
GCGGGATTCCAGGCCTAACACATGCAAGTC
GGCCGTCGACGGTTACCTTGTTACGACTT
GCGGGATTCTTCTTCCGGTTGATCCYGCCGGA
GGCCGTCGACYCCGGCGTTGAMTCCAATT
GCGGGATTCAACCTGGTTGATCCTGCCAGT
GGCCGTCGACTGATCCTTCTGGTTCACCTAC
Table 10 First Sets of Primers Ordered (Delong 1992 PNAS 89:5685-5689). Nucleotides in red
represent BamH 1 sites and nucleotides in green represent Sal 1 sites.
Specific Domain
Eubacteria 2a
Eubacteria 2b
Archaebacteria 2a
Archaebacteria 2b
Eukarya 2a
Eukarya 2b
Primer Name
27FB
1492RS
21FB
958RS
Euk FB
Euk RS
Primer Sequence (5’ to 3’)
GCGGGATCCAGAGTTTGATCCTGGCTCAG
GGCCGTCGACGGTTACCTTGTTACGACTT
GCGGGATCCTTCCGGTTGATCCYGCCGGA
GGCCGTCGACYCCGGCGTTGAMTCCAATT
GCGGGATCCAACCTGGTTGATCCTGCCAGT
GGCCGTCGACTGATCCTTCTGCAGGTTCACCTAC
Table 11 Concentrations and Dilutions of First Sets of Stock Primers. When primers were sent 1
ml of PCR H2O was added and then the primers were diluted to a 10 mM concentration.
Primer Name
Concentration
Eub 27FB 2a
Eub 1492RS 2b
Arch 21FB 2a
Arch 958RS 2b
Euk FB 2a
Euk RS 2b
33.6 nmol
37.9 nmol
53.0 nmol
59.7 nmol
41.2 nmol
36.9 nmol
Amount of Stock
Added
10 µl
10 µl
10 µl
10 µl
10 µl
10 µl
37
Amount of PCR H2O Added to
Make 10 mM Concentration
23.6 µl
27.9 µl
43.0 µl
49.7 µl
31.2 µl
26.9 µl
Table 12 Second Sets of Primers Ordered (Delong 1992 PNAS 89:5685-5689). Nucleotides in
red represent BamH1 sites.
Specific Domain
Eubacteria 3a
Eubacteria 3b
Archaebacteria 3a
Archaebacteria 3b
Eukarya 3a
Eukarya 3b
Primer Name
27FB
1492RS
21FB
958RS
Euk FB
Euk RS
Primer Sequence
GCGGGATCCAGAGTTTGATCCTGGCTCAG
GCGGGATCCGGTTACCTTGTTACGACTT
GCGGGATCCTTCCGGTTGATCCYGCCGGA
GCGGGATCCYCCGGCGTTGAMTCCAATT
GCGGGATCCAACCTGGTTGATCCTGCCAGT
GCGGGATCCTGATCCTTCTGCAGGTTCACCTAC
Table 13 Concentrations and Dilutions of Second Sets of Stock Primers. When primers were
sent 1 ml of PCR H2O was added and then the primers were diluted.
Primer Name
Concentration
Eub 27FB 3a
Eub 1492RS 3b
Arch 21FB 3a
Arch 958RS 3b
Euk FB 3a
Euk RS 3b
70.65 nmol
67.81 nmol
59.94 nmol
79.17 nmol
71.61 nmol
82.16 nmol
Amount of Stock
Added
10 µl
10 µl
10 µl
10 µl
10 µl
10 µl
Amount of PCR H2O Added to
Make 10 mM Concentration
60.7 µl
57.8 µl
49.9 µl
69.2 µl
61.6 µl
72.2 µl
PCR was performed using 12.5 μl ReadyMixTM Taq PCR Reaction Mix with MgCl2
(Sigma Aldrich), 5.5 μl PCR water, 5 μl DNA, and 1 μl each of FB and RS (forward and reverse)
primers from each of the three phylogenetic domains (Eubacteria, Archaea, and Eukarya). These
reactions were mixed and placed in the thermocycler according to the cycles first in Table 14 and
then in Table 15. The samples were then placed into a 1% agarose gel with ExACTGene DNA
ladder as a molecular weight standard and run for 45 minutes at 104 volts. The gels were stained
in 200-300 ml 1X TBE with 10 µl EtBr for 20 minutes. They were viewed under the
transilluminator and photographed using the program LabWorks 4.0. Fifty-seven PCR reactions
were performed on DNA from Extractions #2-5.
38
Table 14 Initial Sets of PCR Cycles (Joplin 1998)
94°C/5 min
94°C/1 min
94°C/1 min
72°C/5 min
50°C/2 min
55°C/1 min
4°C/park
65°C/2 min
72°C/1 min
5 cycles
35 cycles
Table 15 Second Sets of PCR Cycles (Ueda et. al. 1995)
94° C/5 min
94° C/30 sec
94° C/30 sec
72° C/5 min
35° C/1 min
50° C/30 sec
4° C/Park
65° C/2 min
72° C/1 min
5 Cycles
30 Cycles
A gradient was also run to determine which annealing temperature was ideal for each of
the primers (Eubacterial, Archaeal, Eukaryotic). Annealing temperatures for each of the three
phylogenetic domains are displayed below in Table 16. The gradient set up was set up according
to Table 17.
Table 16 Primer Annealing Temperatures
Eubacteria
Archaebacteria
Eukarya
FB
79.7° C
84.3° C
81.0° C
RS
85.4° C
82.8° C
78.6° C
Table 17 Annealing Temperature Gradient Set Up for PCR. Gradient ranged from 78-86°C.
Eubacteria
Archaeabacteria
Eukarya
Well 5
Well 6
Well 10
Well 11
79.3° C
80.5° C
85.1° C
83.8° C
Well 7
Well 8
Well 9
Well 10
81.8° C
83.0° C
84.2° C
85.1° C
Well 4
Well 5
Well 6
Well 7
78.2° C
79.3° C
80.5° C
81.8° C
39
Each of these samples contained 2 µl template DNA, 12.5 µl ReadyMixTM Taq PCR
Reaction mix, 8.5 µl dH2O, and 1µl of each type of primer. The PCR cycles were the following
as shown in Table 18.
Table 18 PCR Cycles for Gradient
95°C/2 min
94°C/1 min
72°C/5 min
82°C/2 min
82°C/1 min
30 cycles
3 cycles
Three 1% agarose gels were prepared and 2 of the gels were run with the PCR products
from the gradient. EtBr (2 µl) (10 µg/µl) and blue juice (2 µl) were added to each of the
samples.
Once bands were obtained from these gels, they were isolated by being cut out with a
sterile razor blade and placed in a spin column in a microcentrifuge tube. This was centrifuged
for 10 minutes at 12,000 rpm. Two and a half volumes of ice cold 95% EtOH and one-half
volume 7.5 M NH4oAc were added to the remaining liquid and this was stored in the freezer
overnight. The next day the tube was spun down for 20 minutes at 14,000 rpm. The supernatant
was poured off and 20 µl DIUF water was added. This isolated DNA was then stored in the
freezer at -20° C.
Cloning
Digest Reactions
Digest reactions were performed with primers from Table 12 using 3 µl PCR product
(Eub), 5 µl 28i LITMUS vector, 2 µl Buffer E, and 1 µl BamH 1 enzyme. This mixture was
40
incubated at 37°C overnight. For plasmids with primers from Table 10, 3 µl PCR product was
used with 5 µl 38i LITMUS vector, 2 µl Buffer E, 1 µl BamH 1 enzyme, 2 µl Buffer D, and 1 µl
Sal 1 enzyme. This mixture was incubated at 37° C overnight. These enzymes were disabled by
adding 10 µl phenol and 10 µl chloroform to each digest reaction. The tubes were spun in the
tabletop centrifuge at 10,000 rpm for 10 minutes. The aqueous layer was removed from each of
the tubes, placed into a new 0.5 ml tube, and 20 µl chloroform was added. This mixture was
spun at 10,000 rpm for 10 minutes and the aqueous layer again removed and placed into a new
0.5 ml microcentrifuge tube. These digest reactions were then used for ligation reactions.
Ligation
Different ligation reactions were used. Ligation protocol #1 (Qiagen (Valencia, CA))
was followed according to the manufacturer’s instructions. This involved 1 µl pDrive cloning
vector, 4 µl of each of the isolated PCR products, and 5 µl Ligation Master Mix (2x). This was
mixed gently and placed in the refrigerator for an hour and stored in the freezer overnight.
Ligation protocol #2 (Promega Corporation (Madison, WI)) was used with a standard (Eub,
Arch, Euk), a positive control, and a background control. For the standard reaction, 5 µl 2x
Rapid Ligation Buffer was used with 1 µl pGEM®-T vector, 3 µl PCR product, and 1µl T4 DNA
ligase (3 Weiss units/µl) for a total of 10 µl. For the positive control, 5 µl 2x Rapid Ligation
Buffer, 1 µl pGEM®-T vector, 2 µl control insert DNA, 1 µl T4 DNA ligase, and 1 µl PCR water
was used. For the background control, 5 µl 2x Rapid Ligation Buffer, 1 µl pGEM®-T vector, 1
µl T4 DNA ligase, and 3 µl PCR water was used. These five reactions were mixed by pipetting,
incubated at room temperature for an hour, and stored in the freezer. For Ligation reaction #3, 4
µl of each PCR product was used (Eub, Arch, Euk) with 1 µl Salt Solution, and 1 µl TOPO
41
Vector (TOPO). This mixture was incubated at 22-23°C for 30 minutes. The reactions were
placed in the freezer at -20° C. Ligation reaction #4 was also performed using Quick Stick
Ligation Kit (Bioline). For this reaction, 4 µl digest product was used along with 10 µl DNA
dilution buffer, 5 µl 4x Qs, and 1 µl QS DNA ligase. This mixture was incubated 5 minutes at
room temperature and placed in the freezer at -20º C.
Transformation
Different transformation reactions were performed. Transformation reaction #1 involved
adding 2 µl of each TOPO ligation reaction to a vial of Invitrogen Mach1-T1 chemically
competent E. coli cells. These were incubated on ice for 30 minutes and heat shocked for 30
seconds in a heating block at 42°C. The vials were transferred to ice and 250 µl room
temperature SOC medium was added to each vial. The tubes were shaken at 200 rpm in an
incubator/shaker for 1 hour. After this incubation/shaking step, 50 µl of each transformation
mixture was added to a prewarmed agar plate with 30 µl X-gal (20 µg/µl). These plates were
placed in an incubator at 37° C overnight. A control reaction was done with 4 µl dH2O, 1 µl Salt
Solution, and 1 µl pCR4-TOPO. This mixture was incubated 5 minutes at room temperature
then placed on ice and then 2 µl of this reaction was added to competent cells. Transformation
reaction #2 was performed using three 1.5 ml tubes of Invitrogen chemically competent Mach1T1 E. coli cells. These cells were thawed on ice for 5 minutes before 2 µl of standard ligation
reaction was added to them. After they were flicked gently, 50 µl of the competent cells were
transferred to tubes with ligation reactions. These tubes were gently flicked and placed on ice
for 20 minutes. The cells were heat shocked for 47 seconds at 42° C and returned to ice for 1520 minutes, and then 950 µl SOC medium (room temperature) was added to each tube. The
tubes were placed for 1.5 hours in the shaking incubator at 37° C at 150 rpm. After incubation,
42
100 µl of each mixture (Eub, Arch, Euk) was added to an agar plate and the plates incubated
overnight at 37° C. For Transformation reaction #3, one tube of competent cells (~200 µl) per
phylogenetic domain (Eubacteria, Archaea, Eukarya) was thawed on ice 35 minutes. Next, 5 µl
of each ligation reaction (Eub, Arch, Euk) was added to each of the tubes with the thawed cells
and was mixed by flicking gently. Each of the tubes was heat shocked at 42° C for 30 seconds.
The tubes were incubated on ice for 2 minutes and 250 µl room temperature SOC medium was
added to each tube. Finally, 100 µl from each transformation mixture was placed on an agar
plate with ampicillin and incubated at 37° C overnight. Transformation reaction #4 involved one
vial of Invitrogen Chemically Competent E. coli cells being thawed on ice and 5 µl ligation
reaction was added very carefully. This was mixed gently by flicking the tube and then
incubated on ice 25 minutes. The mixture was heat shocked at 42° C for 30 seconds in a heating
block. After returning the tube to ice, 250 µl SOC medium was added and the tube placed in the
incubator/shaker at 37° C/200 rpm for 1 hour. After incubation/shaking, the mixture was placed
on three prewarmed plates with 30 µl X-gal (20 µg/µl) each.
Preparation of Tubes for -80° C Freezer
To prepare tubes for storing clones at -80° C, LB broth powder (22.5 g) was added to 900
dH2O and the mixture divided in half. One-tenth volume glycerol (50 ml) was added to the 450
ml volume. One ml was placed in each of one Fisherbrand Screwcap Microcentrifuge tubes
(200) and these were autoclaved for 25 minutes. Once autoclaved, the tubes were stored at 4° C.
Preparation of Stock Plates with Clones
The white colonies that grew from LB transformation plates (Eubacterial, Archaeal, and
Eukaryotic) were made into stock plates by streaking individual colonies on LB plates with
43
ampicillin (25 µg/µl ) and X-gal (10 µg/µl). X-gal indicates whether the insert DNA (in this case
it was the Eubacterial, Archaeal, or Eukaryotic 16s/18s ribosomal gene) was successfully ligated
into the plasmid. The plasmid vectors used in this experiment contain genes for the enzyme βgalactosidase that metabolizes lactose. When lactose is present the bacteria will metabolize the
sugar and produce a compound that combines with X-gal to produces a blue color—causing the
colony to appear blue. However, if the gene sequence is successfully ligated into the plasmid,
the gene for β-galactosidase is disrupted and the bacteria cannot metabolize lactose. This causes
the colony to appear white. The stock colonies that were white were made into bacterial lawns
that were scraped off using a sterile wire loop, given a number, and placed into 1.5 ml prepared
tubes with LB + 10% glycerol and stored at -80° C. Transformation reactions with clones are
shown in Table 19. Designation of clones is shown in Table 20.
Table 19 Numbers of Digest, Ligation, and Transformation Reactions Performed
Digest Reactions
5
Ligations
12
Transformations
21
Table 20 Designation of Clones.
Clones Streaked for
Stock
Put in -80° C for
Storing
Minipreped
Check for
Insert
Eubacteria
302
252
31
26
Archaea
395
17
62
17
Eukarya
132
140
28
14
44
Isolation of Plasmid DNA from Clones
To prepare bacterial clones for plasmid DNA isolation, the clones were briefly thawed.
Bacteria were scraped from these tubes and placed onto warm, LB agar plates with ampicillin
(25 µg/µl ), 30 µl X-gal (10 µg/µl), and incubated at 37° C. An individual colony was taken
from this plate with a sterile wire inoculation loop and placed into an 18 ml glass test tube
containing 1 ml LB broth with 1 µl ampicillin (25 µg/µl). The bacteria were incubated overnight
at 37° C in the shaking incubator (200 rpm). Plasmid DNA isolation (miniprep (Qiagen)) was
performed according to the manufacturer’s instructions. Eluted DNA was stored at -20º C.
All the miniprep reactions were combined according to phylogenetic domain (Eub, Arch,
and Euk). They were purified using 2.5 volumes of 95% EtOH and 0.5 volume NH4oAc.
Volumes are displayed in Table 21. Once these solutions were added to each tube they were
stored at -20° C about 4 hours and then they were taken out and centrifuged for 10 minutes at
10,000 rpm. The supernatants were then poured out and 100 µl Elution Buffer (from Qiagen
Qiaprep® Spin Miniprep Kit) was added.
Table 21 Volumes of Liquid Recovered and Amounts of EtOH and NH4oAc Added
EUB
ARCH 1-8 (each)
EUK 1
EUK 2
Volume
Recovered
300 µl
400 µl
400 µl
300 µl
Volume EtOH Added
750 µl
1000 µl
1000 µl
750 µl
Volume NH4oAc
Added
150 µl
200 µl
200 µl
150 µl
Total
Volume
1200 µl
1600 µl
1600 µl
1200 µl
Insert Confirmation
To confirm that the insert was present in clones, a PCR reaction was run using 5 µl
isolated plasmid DNA (from Qiagen miniprep), 1 µl of each FB and RS primer (Eubacterial with
45
BamH1 sites), 12.5 µl ReadyMixTM Taq PCR Reaction mix, and 5.5 µl water. This PCR reaction
was run under the following conditions shown in Table 22. When the PCR reaction was
complete, the products were checked on a 1% agarose gel.
Table 22 PCR Cycles for Insert Confirmation (Joplin 1998)
94°C/5 min
94°C/1 min
94°C/1 min
72°C/5 min
50°C/2 min
55°C/1 min
4°C/park
65°C/2 min
72°C/1 min
5 cycles
35 cycles
Control colonies also had to be obtained to show that the insert was not present in them.
Individual blue colonies (insert not present) from Eubacterial, Archaeal, and Eukaryotic
transformation reactions were obtained and grown in test tubes 1 ml LB broth with 1 µl
ampicillin (25 µg/µl). These tubes were labeled and placed in the shaking incubator at 150 rpm
at 37° C. Qiagen QIAprep® Spin Miniprep reactions were performed on these controls to isolate
the plasmids. These plasmids were stored in 1.5 ml microcentrifuge tubes at -20° C. Table 23
shows the clones that the plasmid isolation reactions were performed on.
Table 23 Clones Checked for Insert
Eubacteria
Archaea
Eukarya
Clones Checked
Control, 002, 003, 006, 008-010, 012-015
Control, 001, 003-015
Control, 001-010
Control BamH 1 Enzyme Digest
To determine whether the BamH 1 enzyme was working properly a control plasmid with
an 800 bp insert that could be digested out with BamH 1 was obtained. This plasmid was pETt
46
and for this specific reaction 4 µl of the plasmid was used. For the digest reaction, 2 µl pETt
plasmid was mixed with 0.4 µl BamH 1 enzyme, 0.8 µl Buffer E, and 4.8 µl PCR water to bring
the total volume to 8 µl. This reaction was mixed and then placed in the incubator at 37° C
overnight. The digested plasmid as well as 2 µl uncut plasmid were run on a 1% agarose gel.
The samples were loaded into the gel (10 µl of ExACTGene ladder were used as a molecular
weight marker) and the gel was run at 102 volts for 30-45 minutes. The gel was stained in 200
ml 1X TBE with 10 µl EtBr (10 µg/µl).
For the PCR reactions to check for the insert with the transformed bacterial colonies
from above, four new 1% agarose gels were prepared. Archaeal and Eukaryotic clones were
used and twenty-two 0.5 ml microcentrifuge tubes were obtained and labeled: for Archaea C
(control), 1, 3-15 and for Eukarya: C (control), 1-10. For the PCR reactions, 5 µl of each clone
plasmid DNA was placed into the correctly labeled tube and mixed with 12.5 µl ReadyMixTM
Taq PCR Reaction mix, 1 µl of each correct FB and RS primer (Arch or Euk), and 5.5 µl PCR
water. These reactions were mixed by pipetting and then placed into the Eppendorf
thermocycler and run with the same cycles as Table 15. The samples were loaded into the 1%
agarose gels. On two of the gels with the Archaeal samples, 10 µl of the ExACTGene DNA
ladder was used. These gels were run at 102 volts for 45 minutes. On the other two gels with
the Eukaryotic samples, 10 µl of λDNA-HindIII/øx-HaeIII (F-303XSD) DNA molecular weight
marker was used. These gels were run at 104 volts for 30 minutes. The gels were stained in 200
ml 1X TBE buffer with 10 µl EtBr (10 µg/µl ) for 30 minutes. They were viewed on the
transilluminator and photographed.
47
Sequencing
Clones were selected to send off to the University of Tennessee, Knoxville for
sequencing as shown in Table 24. They were wrapped in parafilm and sent overnight to the
sequencing lab. The sequences were returned as an e-mail attachment and opened using the
BioEdit program. Once it was established what the actual sequence was (Appendix C), it was
placed into the BLAST website (NCBI), for identification.
Table 24 Clones Sent for Sequencing
Clone
Euk 001
Eub 001
Arch 001
Euk 001
Eub 002
Eub 003
Eub 007
Eub 020
Euk Control
Euk 009
48
CHAPTER 3
RESULTS
Identification of Organisms Cultured Directly from Roaches
Culturing of Organisms
Bacterial organisms were cultured both anaerobically and aerobically by directly
smearing roach gut tissue and feces on agar plates. The aerobically cultured organisms were
grown on both nutrient and blood agar plates in a candle jar that raised the CO2 levels. The
anaerobically cultured organisms were grown on both nutrient and blood agar in a glass chamber
with a NaBH4 gas pack that took the O2 out of the air. Many colonies were present from these
culture conditions. Colonies were present on all eight of the plates showing typical colony
morphologies. In Figures 4A-D and 5A-D all eight of these plates are present with their bacterial
colonies.
A
B
Figure 4A-B Bacterial Colonies Grown from Roach Feces. Figures A and B organisms were
grown on blood agar. Figure A organisms were grown in the candle jar while Figure B
organisms were grown in the anaerobic chamber.
49
C
D
Figure 4C-D Bacterial Colonies Grown from Roach Feces. Figures C and D organisms were
grown on nutrient agar. Figure C organisms were grown in the candle jar. Figure D organisms
were grown in the anaerobic chamber.
A
B
C
D
Figure 5 Bacterial Colonies Grown from Roach Gut Tissue. In Figures A and B organisms were
grown on blood agar. In Figures C and D organisms were grown on nutrient agar. In Figures A
and C organisms were grown in the candle jar. In Figures B and D organisms were grown in the
anaerobic chamber.
50
Single Colony PCR
When single colonies were chosen off the plates for single colony PCR with Eubacterial
primers bands were present in 8 out of 10 samples. Each of these bands represents the
Eubacterial 16s ribosomal gene 1500 bp long. Figures 6A-B show PCR results from these
reactions.
A
B
1
1500 bp
2
3 4
5
6
7 8
9 10
1
2
3
4
5 6
7 8
9 10
1500 bp
Figure 6 Single Colony PCR Results for Ten Bacterial Colonies. Figure A represents Samples
1-5. (Lanes 2-6). All positive reactions were 1500 bp. Figure B represents Samples 6-10 (Lanes
2-6). No results were seen with samples 2 and 10.
After purification of the DNA, reamplification results were seen for all samples (Figures
7A-B).
Chromosomal DNA Isolation
Chromosomal DNA isolated from bacteria scraped off the plates was very viscous. After
being run on a gel, the anaerobic chromosomal DNA and candle jar chromosomal DNA was run
51
on a gel with the purified single colony PCR samples. The chromosomal DNA yielded results
also shown in Figures 7A-B.
A
B
1 2 3
4 5
6
7
8
9 10
1 2 3 4
5
6
7 8 9 10
Chromosomal
DNA (Lanes
#9 and #10)
1500 bp
1500 bp
Figure 7 Backup PCR and Chromosomal DNA. Figures A and B represent backup PCR with the
ten individual colonies. All ten samples showed bands 1500 bp which is the size of Eubacterial
16s ribosomal gene. In Figure A, Lanes 2-6 represent backup PCR samples 1-5. In Figure B,
Lanes 2-6 represent backup PCR samples 6-10. Lanes 9 and 10 represent isolated chromosomal
DNA from the bacteria scraped from the plates.
PCR from Combined Bacterial Colony DNA
The leftover chromosomal DNA (unspooled) was used for PCR amplification for all three
phylogenetic domains (Eubacteria, Archaea, Eukarya), the chromosomal DNA was diluted
1/5000, and PCR reactions were run for all three phylogenetic domains. The resulting bands are
shown in Figure 8. These products were purified using 95% EtOH and NH4oAc.
52
Eub
Sp
Chr
1
2
Euk
Sp
Chr
3
4
Eub
Unsp
Chr
5
6
7
8
9 10
2000 bp
1500 bp
1500 bp
Figure 8 PCR Results from Bacterial DNA Isolation. The Eubacterial spooled chromosomal
DNA was the streaked band in Lane #2 and it was 1500 bp, the Eubacterial unspooled
chromosomal band was in Lane #5 and was a clean band around 1500 bp, and the Eukaryotic
spooled chromosomal DNA band was in Lane #4 and was a clean band around 2000 bp. 2000
bp is the size of the Eukaryotic 18s ribosomal gene.
When the PCR products (spooled and unspoiled chromosomal DNA) were purified there
were still bands present. Backup PCR reactions for the Eukaryotic samples (Euk Spooled Chr
and Euk Unspooled Chr) yielded 2000 bp bands (size of Eukaryotic 18s ribosomal gene).
Ligation, Transformation, and Plasmid Isolation with PCR Products
There were few white colonies present on the plates that had not been diluted (100
concentration) after ligation and transformation. There were 32 white and blue colonies that
were inoculated in the broth and only 11 that grew. These 11 included 2 Eubacterial and
Eukaryotic controls (blue colonies that had been grown for a negative control), 6 Eubacterial
rDNA samples, and 3 Eukaryotic rDNA samples.
53
Verification of Insert
After the double digest reaction (BamH 1 and Sal 1 enzymes) on the pGEM-T easy
vector (Eukaryotic samples #1-3), there were 2000 bp bands present for all three (Figures 9A-B).
This 2000 bp band represents the Eukaryotic 18s ribosomal gene. The PCR reaction for the Euk
samples showed bands around 2000 bp in all four lanes including the control.
A
B
Control 1
2
3
Control 1
2
3
Plasmid
2000 bp
2000 bp
Figure 9 Results of Eukaryotic Digest and PCR. Figure A represents the digest using BamH 1
and Sal 1 enzymes. The last band present in Lanes #3-5 is around 2000 bp. The Control in Lane
#2 does not show an insert. Figure B represents PCR products and every one of the samples
contains a band around 2000 bp.
The digest with Eubacterial samples #1-10 showed single high molecular weight bands
that suggested there was no 1500 bp (size of Eubacterial 16s ribosomal gene) insert present. The
Eubacterial insert verification PCR reaction yielded 4 1500 bp bands; however, these results are
inconclusive because there was no insert present in the digest reaction.
54
Sequencing and BLAST Results
Sequences obtained from UT Knoxville’s sequencing lab are in Appendix C. BLAST
results for Eubacterial samples #1-10 and Eukaryotic samples #1-3 are shown in Table 25.
Phylogenetic trees created from BLAST are shown in Figures 10-16. The trees for Eubacterial
Samples #2-3, 6, 9 and Eukaryotic Samples #2-3 are in Appendix D.
Table 25 BLAST Results for Samples
EUB 1
EUB 2
EUB 3
EUB 4
EUB 5
EUB 6
EUB 7
EUB 8
EUB 9
EUB 10
EUK 1
EUK 2
EUK 3
Closely related to genus Enterococcus
Closely related to genus Enterococcus
Closely related to genus Enterococcus
Closely related to genus Klebsiella
Member of genus Pseudomonas
Closely related to genus Enterococcus
Closely related to uncultured bacteria found in
chemical treatment plants
Member of genus Fusobacterium
Closely related to the genus Klebsiella
Closely related to genus Serratia
Closely related to genus Gromphadorhina*
Related to genus Gromphadorhina*
Related to genus Gromphadorhina*
* These three samples were actually roach DNA that had been cloned into a vector and
sequenced.
55
Figure 10 Eubacteria Sample #1 Results of BLAST Phylogenetic Tree Analysis. The yellow
highlighted mark labeled lcl|1_4343 represents the sample. This organism is closely related to
the genus Enterococcus.
56
Figure 11 Eubacteria Sample #4 Results of BLAST Phylogenetic Tree Analysis. The yellow
highlighted mark labeled lcl|1_29957 represents the sample. This organism is closely related to
the genus Klebsiella.
57
Figure 12 Eubacteria Sample #5 Results of BLAST Phylogenetic Tree Analysis. The yellow
highlighted mark labeled lcl|1_6346 represents the sample. This organism is a member of the
genus Pseudomonas.
58
Figure 13 Eubacteria Sample #7 Results of BLAST Phylogenetic Tree Analysis. The yellow
highlighted mark labeled lcl|1_19068 represents the sample. This organism is an uncultured
bacterium related to bacteria from a treatment plant.
59
Figure 14 Eubacteria Sample #8 Results of BLAST Phylogenetic Tree Analysis. The yellow
highlighted mark labeled lcl|1_26875 represents the sample. This organism is a member of the
genus Fusobacterium.
60
Figure 15 Eubacteria Sample #10 Results of BLAST Phylogenetic Tree Analysis. The yellow
highlighted mark labeled lcl|1_17150 represents the sample. This organism is closely related to
the genus Serratia.
Figure 16 Eukaryotic Sample #1 Results of BLAST Phylogenetic Tree Analysis. The yellow
highlighted mark labeled lcl|1_29257 represents the sample. The sample is roach DNA because
the results here show that the sequence is within the genus Gromphadorhina.
61
ClustalW Results
Figure 17 shows Eubacteria sample #1 aligned with sequences it is related to.
E.durans
E.faeciumhoneybee
Unculturedmw8
Unculturedmw6
E.villorum
E.ratti
E.hiraeC17456
E.azikeevi
UnculturedOTU9
E.hirae
E.faecium
E.lactis
E.sanguinicola
Unculturedpeh55
E.phoeniculoca
Unculturedmkel
1FB
E.species4fireant
E.durans
E.faeciumhoneybee
Unculturedmw8
Unculturedmw6
E.villorum
E.ratti
E.hiraeC17456
E.azikeevi
UnculturedOTU9
E.hirae
E.faecium
E.lactis
E.sanguinicola
Unculturedpeh55
E.phoeniculoca
Unculturedmkel
1FB
E.species4fireant
E.durans
E.faeciumhoneybee
Unculturedmw8
Unculturedmw6
E.villorum
E.ratti
E.hiraeC17456
E.azikeevi
UnculturedOTU9
E.hirae
E.faecium
E.lactis
E.sanguinicola
Unculturedpeh55
E.phoeniculoca
Unculturedmkel
1FB
E.species4fireant
GGACGAACGCTGGCGGCGTGCCTAATACATGCA 52
GGACGAACGCTGGCGGCGTGCCTAATACATGCA 60
GGACGAACGCTGGCGGCGTGCCTAATACATGCA 52
GGACGAACGCTGGCGGCGTGCCTAATACATGCA 52
GGACGAACGCTGGCGGCGTGCCTAATACATGCA 40
-GACGAACGCTGGCGGCGTGCCTAATACATGCA 32
GGACGAACGCTGGCGGCGTGCCTAATACATGCA 33
GGACGAACGCTGGCGGCGTGCCTAATACATGCA 53
----------------------TAATACATGCA 11
GGACGAACGCTGGCGGCGTGCCTAATACATGCA 42
-GACGAACGCTGGCGGCGTGCCTAATACATGCA 32
GGACGTACGCTGGCGGCGTGCCTAATACATGCA 47
-GACGAACGCTGGCGGCGTGCCTAATACATGCA 32
-GACGAACGCTGGCGGCGTGCCTAATACATGCA 32
GGACGAACGCTGGCGGCGTGCCTAATACATGCA 39
-GACGAACGCTGGCGGCGTGCCTAATACATGCA 32
TCGANGACGCTGGCGGCGTGCCTAATACATGCA 33
CCCCAATCATC--TATCCCACCTTAGGCGGCTG 33
* * *
AGTCGTACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAAGAGTGGCGAACG
AGTCGTACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACG
AGTCGAACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACG
AGTCGAACGCTTCTTTTTCCACCGGAGCTTGCTCCATCGGAAAAAGAGGAGTGGCGAACG
AGTCGAACGCTTCTTTTTCCANCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACG
AGTCGAACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAAGAGTGGCGAACG
AGTCGAACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACG
AGTCGAACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACG
AGTCGAACGCCTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACG
AGTCGAACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAGGAGTGGCGAACG
AGTCGTACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAGGAAGAGTGGCGAACG
AGTCGTACGCTTCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAAGAGTGGCGAACG
AGTCGTACGCTTTTTCTTTCACCGGAGCTTGCTCCACCGAAAGAAAAGGAGTGGCGAACG
AGTCGTACGCTTTTTCTTTCACCGGAGCTTGCTCCACCGAAAGAAAAGGAGTGGCGAACG
AGTCGAACGCTTTTTCTTTCACCGGAGCTTGCTCCACCGAAAGAAAAAGAGTGGCGGACG
AGTCGAACGCTTCTTTTCCACCGAGTGCTTGCACTCATCGGGAAAGAGGAGTGGCGGACG
AGTCGAACGCTTCTT-TCCCACCCCAGCTTGCTCCACCGGGA-AAGAAGAGTGGCGAACG
GCTCCAAAGGTTACCTCACCGACTTCGGGTGTTACA--AACTTTCGTGGTGTGACGGGCG
** * * *
* **
* *** ** **
G-GTGAGTAACACGTGGGTAACCTACCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGAATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTACCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGG-GTGAGTAACACGTGGGTAACCTGCCCACCAGAGGGGGATAACACTTGGAAACAGGTGGTGTGTACAAGGCCCGGG-AACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGA
* ***
** * *** *** *
** *
*
* * * *
** * *
112
120
112
112
100
92
93
113
71
102
92
107
92
92
99
92
91
91
170
178
170
170
158
150
151
171
129
160
150
165
150
150
157
150
149
150
Figure 17 Eubacteria Sample #1 ClustalW Alignment Results. Figure 17 continued.
62
E.durans
E.faeciumhoneybee
Unculturedmw8
Unculturedmw6
E.villorum
E.ratti
E.hiraeC17456
E.azikeevi
UnculturedOTU9
E.hirae
E.faecium
E.lactis
E.sanguinicola
Unculturedpeh55
E.phoeniculoca
Unculturedmkel
1FB
E.species4fireant
--CTAATACCGTATAA-CAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGT
--CTAATACCGTATAA-CAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGAGT
--CTAATACCGTATAA-TAATTAAAACCGCATGGTTTTAATTTGAAAGGCGCTTTACGGT
--CTAATACCGTATAA-TAATTAAAACCGCATGGTTTTAATTTGAAAGGCGCTTTACGGT
--CTAATACCGTATAA-TAATTAAAACCGCATGGTTTTAATTTGAAAGGCGCTTTACGGT
--CTAATACCGTATAA-CAATCAAAACCGCATGGTTTTGATTTGAAAGACGCTTTCGGGT
--CTAATACCGTATAA-CAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGT
--CTAATACCGTATAA-CAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGT
--CTAATACCGTATAA-CAATCGAAACCGCATGGTTTCGATTTGAAAGGCGCTTTCGGGT
--CTAATACCGTATAA-CAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGT
--CTAATACCGTATAA-CAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGT
--CTAATACCGTATAA-CAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGT
--CTAATACCGTATAA-CAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGT
--CTAATACCGTATAA-CAATCGAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCGGGT
--CTAATACCGTATAA-CAATTGGAACCGCATGGTTCTAATTTGAAAGACGCTTTCGGGT
--CTAATACCGTATAA-CAATCGGAACCGCATGGTTCTGATTTGAAAGGCGCTTTCGGGT
--CTAATACCGCATAA-TACATCGGATCTCATGGTCTGATGTTGAAAGGCGCTTTCGGGT
TTCCGGCTTCATGTAGGCGAGTTGCAGCCTACAATCC-GAACTGAGAGAAGCTTTAAGAG
*
*
**
* * *
*
*** ** *****
227
235
227
227
215
207
208
228
186
217
207
222
207
207
214
207
206
209
E.durans
E.faeciumhoneybee
Unculturedmw8
Unculturedmw6
E.villorum
E.ratti
E.hiraeC17456
E.azikeevi
UnculturedOTU9
E.hirae
E.faecium
E.lactis
E.sanguinicola
Unculturedpeh55
E.phoeniculoca
Unculturedmkel
1FB
E.species4fireant
GTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GCCACTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GCCACTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GCCACTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GTCACTGATGGATGGACCTGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GTCACTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
GTCACTGGTGGATGGACCCGCGGTGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGG
ATTA-----GCTTAGCCTCGCGACTTCGCGACTCGTTG-------TACTTCCCATTGTAG
* * * * ***
** ****
** * **
*
287
295
287
287
275
267
268
288
246
277
267
282
267
267
274
267
266
257
E.durans
E.faeciumhoneybee
Unculturedmw8
Unculturedmw6
E.villorum
E.ratti
E.hiraeC17456
E.azikeevi
UnculturedOTU9
E.hirae
E.faecium
E.lactis
E.sanguinicola
Unculturedpeh55
E.phoeniculoca
Unculturedmkel
1FB
E.species4fireant
CTACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CGACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CGACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CGACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CGACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CGACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CGACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CGACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CGACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
CCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCA
CAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCA
CGACGATACATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCA
C-ACG-TGTGTAGCCCA--------GGTCATAAGGGGCATGATGATTTGACGTCATCCCC
* *** *
***** *
*** ** *
**
** *
* ***
347
355
347
347
335
327
328
348
306
337
327
342
327
327
334
327
326
307
E.durans
E.faeciumhoneybee
Unculturedmw8
Unculturedmw6
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
407
415
407
407
E.villorum
E.ratti
E.hiraeC17456
E.azikeevi
UnculturedOTU9
E.hirae
E.faecium
E.lactis
E.sanguinicola
Unculturedpeh55
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
395
387
388
408
366
397
387
402
387
387
Figure 17 continued
63
E.phoeniculoca
Unculturedmkel
1FB
E.species4fireant
GACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
GACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
AACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA
ACCTTCCTCCGG--TTTGTCACCGGCAGTCTT--GCTA-----GAGTGCCCAACTTAATG
** * * **
*
** * **** ** *
** * * ** *
394
387
386
358
E.durans
E.faeciumhoneybee
Unculturedmw8
Unculturedmw6
E.villorum
E.ratti
E.hiraeC17456
E.azikeevi
UnculturedOTU9
E.hirae
E.faecium
E.lactis
E.sanguinicola
Unculturedpeh55
E.phoeniculoca
Unculturedmkel
1FB
E.species4fireant
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGGAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTTAGAGAAGAACAA
ACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTT-GTCAGAGAAGAACAA
ATGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACAC
* * *
* *
*
* *
*
* *
***
*
* ***
466
474
466
466
454
446
447
467
425
456
446
461
446
446
453
446
445
418
E.durans
E.faeciumhoneybee
Unculturedmw8
Unculturedmw6
E.villorum
E.ratti
E.hiraeC17456
E.azikeevi
UnculturedOTU9
E.hirae
E.faecium
E.lactis
E.sanguinicola
Unculturedpeh55
E.phoeniculoca
Unculturedmkel
1FB
E.species4fireant
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCRTCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGA-TGAGAGTAACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GGG-TGAGAGTAACTGTTCATCCTTTGACGGTATCTGACCAGAAAGCCACGGCTAACTAC
GAGCTGACGACAACCATGCACCACCTGTCACTTTGCCCCCGAAGGGGAAGCTCTATCTCT
*
***
*** * * *
** * * *
** * * *
*** **
525
533
525
525
513
505
506
526
484
515
505
520
505
505
512
505
504
478
E.durans
E.faeciumhoneybee
Unculturedmw8
Unculturedmw6
E.villorum
E.ratti
E.hiraeC17456
E.azikeevi
UnculturedOTU9
E.hirae
E.faecium
E.lactis
E.sanguinicola
Unculturedpeh55
E.phoeniculoca
Unculturedmkel
1FB
E.species4fireant
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
AGAGTGGT--CAAAGGATGTCAAGACCTGGTAAG-GTTCTTCGCGTTGCTTCGAATTAAA
*
*
**
*
* *** *** *** * ** ** ** *
****
585
593
585
585
573
565
566
586
544
575
565
580
565
565
572
565
564
535
E.durans
E.faeciumhoneybee
Unculturedmw8
Unculturedmw6
E.villorum
E.ratti
E.hiraeC17456
E.azikeevi
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAAG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
642
650
642
642
630
622
623
643
Figure 17 continued
64
UnculturedOTU9
E.hirae
E.faecium
E.lactis
E.sanguinicola
Unculturedpeh55
E.phoeniculoca
Unculturedmkel
1FB
E.species4fireant
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
GCGAGCGCAG-GCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGG--CTCAACCGGGGAGG
CCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGT
*
**
* * ** *
* *
** ** **
*
****** *
E.durans
E.faeciumhoneybee
Unculturedmw8
Unculturedmw6
E.villorum
E.ratti
E.hiraeC17456
E.azikeevi
UnculturedOTU9
E.hirae
E.faecium
E.lactis
E.sanguinicola
Unculturedpeh55
E.phoeniculoca
Unculturedmkel
1FB
E.species4fireant
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
GTCATTGGAAACTG
CGTACTCCCCAGGC
** *
601
632
622
637
622
622
629
622
621
595
701
709
701
701
689
681
682
702
660
691
681
696
681
681
688
681
635
655
Figure 17 Eubacteria Sample #1 ClustalW Alignment Results. The organisms seen here were
chosen because they are different species that closely related to the sample. The other
Eubacterial samples were compared with similar sequences as well.
65
DNA Isolation and Quantification from Roach Gut Tissue
DNA isolation #1 yielded no discernable DNA band in the gel electrophoresis. DNA
Isolation #2, however, produced DNA with some degradation. DNA isolation #3 produced
DNA; however, it was not very visible and 10 µl had to be used instead of 5 µl. DNA Isolation
#4 yielded bright high molecular weight bands as shown in Figure 18. DNA Isolation #5
produced high molecular weight DNA with impurities present. Quantification results for DNA
Isolations #2-4 are shown in Table 26.
Table 26 Values for DNA Isolation Quantifications
DNA Isolation #2
DNA Isolation #3 (1)
DNA Isolation #3 (2)
DNA Isolation #4
260/280 Ratio
Normal Range
0.00185
0.97700
0.80000
1.66000
1.5-2.0
1.5-2.0
1.5-2.0
1.5-2.0
1 2 3 4 5 6 7 8
2 µl
9 10
5 µl
Figure 18 Total Genomic DNA Isolation #4. The first band represents 5 µl of high molecular
weight DNA and the second band represents 10 µl of high molecular weight DNA.
66
When the total genomic E. coli DNA was extracted and run on the gel however, there
was a high molecular weight band present. This is evident in Figure 19.
1
2
3
4
5
6
7
8
9
10
Figure 19 E. coli DNA Extraction Results. The extraction yielded in a large band which shows
high molecular weight chromosomal DNA (Lane 5).
PCR Amplification
Of the 57 PCR reactions that were performed from DNA isolations #2-5, there were 20
positive results and 34 negative results as shown in Table 27. A positive reaction meant that
there was a band of the correct size of the 16s/18s ribosomal gene for a particular phylogenetic
domain (Eubacteria, Archaea, Eukarya).
67
Table 27 Results from All PCR Reactions
Positive Eubacteria
Positive Archaea
Positive Eukarya
Nothing
Other
PCR Reactions
9
5
6
34
3
% of Total Reactions
15.79
8.77
10.53
59.65
5.26
57
100
Total Reactions
One of the most significant PCR results is shown in Figure 20. The Eubacterial bands
were 1400-1500 bp (older primers had smaller bands)(represents 16s ribosomal gene size), the
Archaeal bands were 975 bp (represents 16s ribosomal gene size) and were the same size and the
Eukaryotic bands were around 1900 bp (represents 18s ribosomal gene size) and were roughly
the same size. Following PCR reactions produced Eubacterial bands 1500 bp.
1
Eub Eub
2
3
Arch Arch Euk Euk
4
5
6
7
1500 bp
8
9
10
2000 bp
975 bp
2000
1500
1000
900
Figure 20 PCR Results from Old and New Eubacterial, Archaeal, and Eukaryotic Primers. The
second, fourth, and sixth lane represents PCR bands using the old primers from Table 9 and the
third, fifth, and seventh lanes represent PCR bands using the new primers from Table 10.
68
Cloning
The cloning reactions were done using digests, ligations, and transformations. The
pDrive cloning vectors (Qiagen) yielded no colonies except on the Archaeal plates. The control
reaction also yielded no results (no colonies grew on the plates). When the ligation and
transformation reactions were redone using the pGEM®-T Vector (Promega) and Mach1-T1
chemically competent E. coli cells), white colonies were visible. However, these white colonies
surrounded the present blue colonies and were much smaller, which indicates that these were
satellite colonies. Satellites occur when the bacteria do not take up the plasmid with the
ampicillin thus are unable to grow on the plates. However, the colonies that do grow produce
penicillinase that breaks up the ampicillin and allows the bacteria without the plasmid to grow.
They appear as smaller white colonies surrounding the blue ones. When the TOPO cloning kit
(Invitrogen) as well as their transformation reaction with Mach1-T1 chemically competent E.
coli cells was performed, nothing was present except one Eukaryotic colony. This colony was
made into 8 stock colonies of which 7 grew.
Transformations using the 28i and 38i LITMUS vectors (New England Biolabs) and
Quick Stick Ligation Kit (Bioline Inc), yielded many white colonies or colonies that contained
the ribosomal gene insert. These isolates were streaked onto new plates and numbered to make
them into stock colonies. The digest, ligation, and transformation reactions for the Archaea and
Eukarya also produced white colonies. These were also streaked onto plates to make stock
colonies. The transformation redone for the Archaea yielded mostly blue colonies with white
satellite colonies.
When the transformation was performed after using phenol and chloroform extraction,
white isolates were present. Results from this reaction were successful yielding white colonies.
69
Clones inoculated in LB broth for plasmid isolation, a significant number grew. The results of
these digest/ligation/transformation/plasmid isolation reactions are displayed in Table 28.
Table 28 Results of Digest, Ligation, Transformation, and Plasmid Isolation Reactions
Digests that
Worked
Ligations
that Worked
2
4
2
8
Eubacteria
Archaea
Eukarya
Total
Reactions
4
7
3
Transformations
that Yielded
Colonies
4
7
4
White
Colonies
Present
302
395
132
Number of Colonies
Grown for Plasmid
Isolation
22
81
26
14
15
829
129
Insert Verification
Restriction digests (BamH 1 only and BamH 1 and Sal 1) done to determine whether
inserts (16s/18s ribosomal genes) were present in the Eubacterial, Archaeal, and Eukaryotic
clones were unsuccessful. However, some of the PCR reactions for the Eubacteria and Eukarya
appeared to yield positive results (bands that were the right size for the ribosomal genes for these
two phylogenetic domains) as shown in Figures 21A-B, 22C-D. Figures 22A-B represents PCR
reactions for Archaeal insert verification. Results for the restriction digest and PCR reactions are
shown in Table 29.
Table 29 Results of Restriction Digests and PCR Reactions for Insert Verification
Eubacteria
Archaea
Eukarya
Positive Restriction Digest
Samples
31
1
12
70
Positive PCR Reaction
Samples
0
0
0
A
B
1
2 3
4
5 6 7 8
9 10
1500 bp
1
2 3 4
5
6
7
8
9 10
1500 bp
Figure 21 PCR Results for Eubacterial Insert Verification. Figure A represents Eubacterial
Clones (PCR) Control, 004-008, 011, and 012 in lanes 2-9. Figure B represents Eubacterial
Clones (PCR) 013-015, 020-022, 026, and 028 for insert verification (Lanes 2-9). The control
represents a blue colony that was grown in broth, minipreped, and a PCR reaction done on this as
well. Only Clone 026 in Figure B showed nothing (Lane 8).
A
B
1
2 3 4 5 6 7 8
9 10
1 2 3
4 5
6 7 8
9 10
Figure 22A-B PCR Results for Archaeal and Eukaryotic Insert Verification. Figure A represents
Archaeal Clones (PCR) Control 001, 003, 004, 005, 006, 007, and 008 in Lanes 2-10. Figure B
represents Archaeal Clones (PCR) 009, 010, 011, 012, 014, and 015 in Lanes 2-7.
71
C
D
1
2
3 4
5
6
7 8
9
1
2
3
4
5
6
7
8
9
2000 bp
2000 bp
Figure 22C-D PCR Results for Archaeal and Eukaryotic Insert Verification. Figure C represents
Eukaryotic Clones (PCR) Control, 001-005 in Lanes 2-7. Figure D represents Eukaryotic Clones
(PCR) 006-010 in Lanes 2-6.
Control BamH 1 Reaction
When the BamH1 enzyme was checked for activity using the pETt plasmid, results
demonstrated that the enzyme was still active (Figure 23).
72
Uncut Plasmid
Cut Plasmid
800 bp insert
Figure 23 Digest Results from Control Reaction Checking BamH 1 Activity. Lane #3 shows 800
bp insert and high molecular weight plasmid.
Sequencing
Clones prepared and sent off to the University of Tennessee Knoxville’s sequencing lab
only yielded cloning vector sequences. No gene insert was present in any of the clones as shown
in Table 30. These gene sequences can be found in Appendix C.
Table 30 Results of Clone Sequencing
Clone Name
EUB 001
EUB 002
EUB 003
EUB 007
EUB 020
ARCH 001
EUK Control
EUK 001 (1) T3
EUK 001 (1) T7
EUK 001 (2)
EUK 009
Results
No Significant Similarity Found
No Significant Similarity Found
No Significant Similarity Found
No Significant Similarity Found
Cloning Vector
No Significant Similarity Found
Cloning Vector
RAGE Cloning Vector pRIG20
No Significant Similarity Found
No Significant Similarity Found
No Significant Similarity Found
73
CHAPTER 4
DISCUSSION
Research Applications
The ecology of microorganisms in association with macro organisms is a largely
unexplored area of research. Even today there are technical difficulties in identifying the
diversity of these organisms and this hinders research. Studies have shown that it is possible to
use molecular techniques to determine the diversity of life; however, this approach has also
demonstrated that >90% of species are unknown. In addition, only preliminary studies have
been published on the ecological interaction of this diversity of organisms. Interacting networks
and web association is just beginning to be explained in ecology. The gut ecology would be an
ideal model system since it has been shown that the alimentary canal of organisms harbors
organisms comprising the three phylogenetic domains of life (Cruden and Markovetz 1987). The
ecology of endosymbionts in the alimentary canal of insects is a model of micro ecology that
could be explored. Research has previously demonstrated that there is apparently a vast diversity
of both anaerobic and aerobic bacteria present in the gut of the American cockroach P.
americana and the cave-dwelling roach E. posticus. Many of these are common identified
species such as Enterobacter agglomerans, Klebsiella oxytoca, Citrobacter freundii, Serratia
species, and Streptococcus species. Other organisms frequently isolated were Clostridium
sporogenes, Fusobacterium varium, Eubacterium moniliforme, and Peptococcus variabilis
(Cruden and Markovetz 1987). In addition to anaerobic and aerobic bacteria, protozoans
harboring bacteria or having bacteria associated with them (usually these bacteria are
methanogens) have been isolated. Spirochetes and nematodes have also been identified in the
74
hindgut (Cruden and Markovetz 1987, Gijzen and Barugahare 1992). The goal of my study was
to identify and describe some of the typical endosymbionts involving bacterial species,
archaeabacteria, and eukaryotes in the gut of G. portentosa.
Molecular techniques such as PCR and cloning have allowed scientists to investigate
diversity of organisms without having to resort to traditional techniques such as cultivation on
petri dishes or in broth. For example, scientists investigated the biodiversity of microbial
eukaryotes in the Antarctic Ocean through the use of molecular techniques (PCR) that amplified
SSU rNA sequences. Through the use of PCR amplification many new lineages of picoplankton
and nanoplankton were identified (Moriera and López-García 2002). These PCR and cloning
techniques were applied to total DNA extracted from G. portentosa gut in my experiment in
hopes that a vast array of eukaryotes could be found and identified. The results shown in Figure
20 demonstrate that it is possible to amplify Eukaryotic DNA using domain-specific primers
through PCR.
In addition, molecular techniques have also led researchers to discover that the majority
of bacteria present in the guts of insects are γ-proteobacteria from the γ-3 subdivision of
Proteobacteria. Five genomes from insect endosymbionts have been sequenced, allowing for
better understanding of how symbiosis is established. These secondary bacteria can be found not
only in the gut tissue but also in glands, body fluids, or in cells that surround other
endosymbionts. These bacteria are primarily the results of multiple independent infections (Gil
et al. 2004). It is possible that several of the organisms identified in this study such as had
gained access to G. portentosa’s alimentary canal through independent infections such as
handling, food, and drinking water. The reasoning behind this is that when the organisms were
identified, Eubacterial Samples 7 and 10 were organisms typically found in the environment and
75
would only have entered the roach through the environment. Bacterial organisms that were
identified appeared to belong to these groups of γ-proteobacteria since they belonged to or were
very closely related to the genera Enterococcus, Fusobacterium, Klebsiella, Pseudomonas, and
Serratia.
Isolation and Identification of Microflora from Roach Gut and Feces
The bacterial colonies cultured from the extracted roach gut and feces were cultured both
anaerobically (anaerobic chamber) and aerobically (candle jar). These traditional methods were
used because the digest, ligation, and transformation reactions used previously did not appear to
produce detectable results. BLAST results showed that the previous sequences using molecular
techniques were inconclusive. Single colony PCR amplification however, yielded bright 1500
bp bands that represented the size of the Eubacterial 16s ribosomal gene. BLAST results from
these PCR amplifications, and sequencing demonstrated that these sequences belonged to
bacteria. Eight of the 10 of the samples were typical bacteria found within the gastrointestinal
tract of the roach and belong to five major genera of enteric bacteria. The other two samples can
be found in the environment (#7, 10). Eubacterial samples #1-3, and 6 were very closely related
to the genus Enterococcus. Eubacterial sample #8 was closely related to the genus
Fusobacterium. Eubacterial samples #4 and #9 were very closely related to the genus Klebsiella.
Eubacterial sample #5 was very closely related to the genus Pseudomonas. Eubacterial sample #
10 was very closely related to the genus Serratia. Eubacterial sample #7 was closely related to
uncultured bacterial clones found in soil samples, Pseudomonas, and a hypertrophic freshwater
lake in China. The identity of this organism was unclear.
76
The genus Enterococcus comprises the group of gram-positive cocci (circular-shaped
organisms) that often appear in pairs, alone, or in chains. These bacteria are facultatively
anaerobic and can be found in soil, food, water, plants, animals, humans, birds, etc. When they
inhabit an animal or a human, however, they inhabit the gastrointestinal or genital tract. When
these organisms escape the gastrointestinal tract and inhabit other parts of the body they may
cause bacteremias and urinary tract infections (Facklam et al. 1970). Four out of 10 of the
identified bacterial organisms from the G. portentosa gut were very closely related to this genus
of bacteria. These are what would be the expected bacteria or organisms that would normally be
encountered in an ecological study like this one.
The genus Fusobacterium comprises a group of anaerobic, gram-negative bacilli that are
typically found in the mouth, upper respiratory tract, and intestinal tract of healthy individuals
(Jousimies-Somer et al. 1970). One of the 10 identified bacterial samples was very closely
related to the genus Fusobacterium.
The genus Klebsiella is a group of nonmotile, rod-shaped, gram-negative organisms that
contain a capsule. They are anaerobes that are primarily found in the gastrointestinal tract, skin,
and pharynx of humans but also thrive in the gastrointestinal tract of other organisms as well.
Klebsiellae are often responsible for opportunistic infections in the respiratory tract and may
produce beta-lactamase which is an enzyme that degrades penicillin and other beta lactam
antibacterial drugs (Umeh and Berkowitz 2006). Two of the 10 bacterial samples obtained
through single colony PCR were very closely related to the Klebsiella genus.
The genus Pseudomonas is a group of aerobic non-spore forming, gram-negative slightlycurved rods. These organisms are mobile because of flagella that propel them. These particular
organisms are generally found in most moist environments. These include soil, on plants (fruits
77
and vegetables), and in watery environments such as swimming pools, hot tubs, and contact lens
solutions. Pseudomonas colonizes the gastrointestinal tract of normal individuals and also may
colonize the throat, nasal mucosa, and other moist skin surfaces. These organisms can cause
infections in immunocompromised individuals and those with cystic fibrosis (Kiska and Gilligan
1970). One of the 10 identified bacterial samples was very closely related to this genus.
The genus Serratia is a group of organisms that are gram-negative, facultative anaerobic,
rods, or coccobacilli. These bacteria are widely spread throughout the environment and can
cause infections in the gastrointestinal tract (Abbott 1970). Of the 10 bacterial samples, only one
was very closely related to Serratia.
The three Eukaryotic samples obtained from cloning DNA from cultured organisms from
the roach gut appeared to all be roach DNA. The BLAST results show that these samples are all
closely related to the Madagascar hissing roach Gromphadorhina portentosa and
Gromphadorhina laevigata.
Total DNA Extraction from Roach Tissue
Results from the DNA extractions indicate that it is in fact possible using extraction
methods to extract total genomic DNA from G. portentosa. There were a lot of impurities
present in these DNA samples, however, and the DNA was often degraded, thus there was not a
good, clean band of DNA on the gels. The DNA should have been more carefully extracted by
using phenol and chloroform one after another instead of together. This also indicates that a
more efficient method of total DNA extraction is needed.
78
PCR Amplification
In was established through PCR in Figure 20 that there were organisms comprising all
three phylogenetic domains present in the alimentary canal of G. portentosa. Eubacterial bands
were 1500 bp (16s ribosomal gene), the Archaeal bands were 975 bp (16s ribosomal gene) and
the Eukaryotic bands were 2000 bp (18s ribosomal gene). Each band present in the gel
represents gene sequences from thousands if not millions of different organisms belonging to
each of the three phylogenetic domains (Eubacteria, Archaea, and Eukarya), some of these which
have not been identified yet. These results indicate that the roach gut could serve as a model
ecosystem because it houses organisms comprising the three phylogenetic domains of life.
Cloning and Sequencing
The majority of problems, however, with this study were with the digest, ligation, and
transformation steps. The problems arose when the purified PCR DNA was digested with the
restriction enzymes BamH 1 and Sal 1 and ligated into a vector. One suitable reasoning is that
the enzymes were not working as they should or the wrong buffers were used causing the
inefficiency. During earlier digests with the Eubacterial samples, both enzymes (BamH 1 and
Sal 1) were used along with their specific buffers (Buffers E and D). However, when these same
enzymes were just with just Buffer E (BamH 1 buffer), the insert was digested out. This could
be because previously there was no insert present in the plasmid, or the enzyme was more
efficient with just Buffer E. More reasons for why the ligation step did not work could be
because the vector and insert were in the wrong proportions thus causing insert not to get into the
vector. Another possibility is that the DNA could have been chewed up or degraded and thus not
be able to be ligated into a vector. The transformation reactions faced many problems as well.
79
There were a lot of satellite colonies resulting when a transformation was performed. Another
problem that occurred with the transformation is that in several instances the cells were old and
did not grow. It was determined that the transformation reactions were unsuccessful when
digest and PCR reactions were done to confirm the presence of the insert. None of the clones
showed an insert present and when the PCR amplification reactions were done, they showed
bands for the controls in addition to the regular clones. The majority of these transformation
reactions were unsuccessful and the results inconclusive.
The clones that were selected to send off for sequencing (Table 24) all showed
undetectable results or showed a cloning vector when placed into the BLAST database for
analysis (Table 25). This demonstrates that the gene insert was not present at all. All of these
clones indicate inconclusive results and were not used for ClustalW analysis. The gene
sequences that were successfully ligated into the vector showed up as roach DNA. Even though
the samples were roach DNA they can still be considered part of the model ecosystem because
the host plays a role in the relationship with the endosymbionts.
Future Research
Future investigation to obtain a larger amount of organisms would be to use more
culturing techniques besides just the candle jar and anaerobic chamber with the gas pack. Other
culturing media could be used as well such as McConkey agar and LB agar. The visible
organisms could then be collected, the DNA isolated, and PCR reactions could be done using
more efficient primers such as Eubacterial 63f and 1387r (Marchesi et al. 1998). Another
method would be to perform single colony PCR on as many different colonies as could be
80
detected. The gene sequences obtained from these colonies could then be placed into BLAST
and identified based on sequence similarity and then placed into ClustalW for further analysis.
Further applications with my research could be that once more of organisms present in
the roach gut are identified, they could be studied in closer detail to understand their interactions
with the host organism. Studies with N. ovalis and its indwelling methanogens showed that the
protozoan had a major effect on P. americana metabolism, body weight, and methane production
(Gijzen and Barugahare 1992). These protozoans and methanogens are involved in methane
production, hindgut metabolism, insect body weight, and generation time. When cockroaches
were raised free of N. ovalis insect generation time increased, adult body weight decreased, and
there was no methane production (Gijzen and Barugahare 1992). The knowledge of these
organisms will then give researchers a better understanding of insects and how they interact with
the world around them as well as their importance to the ecosystem.
Another example interaction of host organism/bacterial symbiosis is in honeybees. Four
of particular symbionts contain bacteriostatic effects against the honeybee pathogen
Paenibacillus larvae larvae. A study was done that isolated bacterial species from 7- and 1-dayold honeybee larvae. Twenty-three isolates that inhibited the pathogen were cultured from the
larvae. These isolates included ten Bacillus sp. with an additional seven showing conditional
inhibition. Of these 17 isolates the seven conditional inhibition ones belonged to the Bacillus
cereus group and of the other 10 eight of them were B. cereus and two were B. fusiformis. In
addition, matches with Brevibacillus formosus, isolates tied to Stenotrophomonas maltophilia as
well as two Acinetobacter isolates inhibited the pathogen as well (Evans and Armstrong 2006).
Further research with G. portentosa endosymbionts could demonstrate that these organisms work
together to prevent disease in the roach.
81
Once it is established how the endosymbionts interact with their hosts, scientists can
investigate how the symbionts interact with each other. For example, research has shown that
the enteric organism Escherichia coli is capable of forming a biofilm with the help of other
bacteria. Non-adhering E. coli strains were grown in the presence of other adhering bacterial
species and formed biofilms and co-adhesion mechanisms. Non-adhering E. coli strain PHL565
was mixed with Pseudomonas putida and showed an increase in biofilm production and
adherence to glass. There was an increase in the biomass of cells by four fold and the
composition of the E. coli PHL565 was roughly the same as the E. coli/P. putida composition
from the previous experiment. These experiments demonstrate that bacteria unable to adhere to
surfaces may be enabled by the presence of other bacterial species, thus showing the benefits of
an isolated ecosystem (Castonguay et al. 2006). It is possible that microfloral organisms
inhabiting the G. portentosa alimentary canal could benefit each other in a similar way or even
performing the same function.
Molecular techniques allow the identification of organisms and view of them from an
ecological perspective—the roach alimentary canal is considered an isolated ecological system.
Results support the working hypothesis (stating that organisms comprising all three phylogenetic
domains are present in the roach alimentary canal and these include known as well as unknown
organisms) and enhance previous studies that have been done using traditional techniques
(Cruden and Markovetz 1987). The purpose of this experiment was an ecological study to get an
idea of the number of endosymbionts (from all three phylogenetic domains) in the alimentary
canal of roaches using molecular techniques. Through further research and improved techniques,
it may be possible to identify more specific organisms from all three phylogenetic domains and
further increase knowledge of the world of microbiology.
82
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85
APPENDICES
APPENDIX A: Abbreviations
amp ampicillin
ARCH Archaeal/Archaea
BHI brain heart infusion
BLAST Basic Local Alignment Search Tool
bp base pairs
dH2O deionized water
DIUF deionized ultra filtered
DNA deoxyribonucleic acid
dNTP deoxyribonucleoside triphosphate
DTT dithiothreitol
EDTA ethylenediaminetetraacetic acid
EMBL-EBI European Molecular Biology Laboratory-European Bioinformatics Institute
EtBr ethidium bromide
EtOH ethanol
Eub Eubacterial/Eubacteria
Euk Eukaryotic/Eukarya
IPTG Isopropyl-β-D-thiogalactopyranosid
LB Luria-Bertani
M molar
mg milligram
86
ml milliliter
mM millimolar
NaBH4 sodium borohydride
NCBI National Center for Biotechnology Information
NH4oAc ammonium acetate
PCR polymerase chain reaction
rDNA ribosomal deoxyribonucleic acid
rpm rounds per minute
rRNA ribosomal ribonucleic acid
SDS sodium dodecyl sulfate
TBE tris-boric acid EDTA buffer
TE tris-EDTA buffer
µg microgram
µl microliter
µM micromolar
X-Gal 5-Bromo-4-Chloro-3-Indolyl-Beta-D-Galactopyranoside
87
APPENDIX B: Recipes
1% Agarose Gel
30 ml TBE Buffer
0.03 gram Agarose
Heat for 45 seconds in microwave
Allow to cool 20-30 minutes
Staining Medium
200 ml 1X TBE buffer
10 µl EtBr (10 µg/µl )
10X TBE Buffer
55 g boric acid
9.3 g EDTA
108 g Tris Base
1000 ml distilled water
1X TBE Buffer
100 ml 10X TBE buffer
900 ml distilled water
X-gal Preparation
5 ml N, N-dimethylformamide to bottle of X-gal
88
10X TE Buffer
108 g Tris Base
9.3 g EDTA
For 1X TE add 100 ml 10X TE to 900 ml dH2O
LB Broth
25 grams LB powder
1000 ml dH2O
Autoclaved for 20-25 minutes
Ampicillin (25 mg/ml or 25 µg/µl)
20 ml dH2O
500 mg ampicillin salt solution
LB Plates with Ampicillin (25 µg/µl ) /X-Gal (20 µg/µl )
40 grams Luria-Bertani powder
1000 ml distilled water
Autoclaved 20-25 minutes
Cooled for 45-60 minutes
Ampicillin 25 mg/ml (25 µg/µl ) (1 µl per ml of liquid)
30-40 µl X-Gal (20 µg/µl )
89
Insect Saline
7.5 grams NaCl
0.21 gram CaCl2
0.25 gram KCl
Add to 1000 ml distilled H2O and autoclave 20 minutes
70% EtOH
70 ml 200 Proof (100% EtOH)
30 ml dH2O
90
APPENDIX C: Gene Sequences from BLAST
Eub 1 27 FB
TCGANGACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCTTCTTTCCCACCCC
AGCTTGCTCCACCGGGAAAGAAGAGTGGCGAACGGGTGAGTAACACGTGGGTAACC
TGCCCACCAGAGGGGGATAACACTTGGAAACAGGTGCTAATACCGCATAATACATC
GGATCTCATGGTCTGATGTTGAAAGGCGCTTTCGGGTGTCACTGGTGGATGGACCCG
CGGTGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATACATAGCCG
ACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGG
AGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCG
TGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTCAGAGAAGAACAAGGGTG
AGAGTAACTGTTCATCCTTTGACGGTATCTGACCAGAAAGCCACGGCTAACTACGTG
CCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAA
AGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGA
GGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCCATGT
GTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAAGGCGGCTCTC
TGGTCTGTAACTGACGCTGAGGCTCGAAGCGTGGGGAGCAACAGGATTAGATACCC
TGGTAGTCCCNCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTNCCCCCTTCAGTG
CTGCAGCTAACGCATTAAGCACTCCNCCTGGGGAGTACNACCNCAAGGTTGAAACT
CAAGGAATTGACGGGGNCCNCCNANCCGGTGGAACATGNGGTTTTATTCNAAACAA
CNCGAAAAACTTNNCAGGNCTTGACTNCNTTTGACNCTCTNNAAAAANNGCTTTCCC
TTCGGGGANAANTGANNGGNGGNGCTTGNTTNCCNCACTCCNTTCTGANATTTNGGT
TAATCCCCAACAGCCACCCTTTTTTNNTGCCNCTTCATTGGGNCCTNNNGNCNGCGG
NNAACCGGGAGGG
Eub 1 1492RS
CCCTATCTCTGTCCTACCTTAGGCGGCTGGCTCCATAAATGGTTACCCCACCNACTTC
GGGTATTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTA
TTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGCAGGCGAGT
TGCAGCCTGCAATCCGAACTGAGAAAGGTTTTAAGAGATTCGCTTGACCTCGCGGTC
TTGCTGCTCGTTGTACCTTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCA
TGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCGCTAGAG
TGCCCAACTGAATGATGGCAACTAGCAATAAGGGTTGCGCTCGTTGCGGGACTTAAC
CCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTTTGTCCCC
GAAGGGAAAGCTCTATCTCTAGAGTGGTCAAAGGATGTCAAGACCTGGTAAGGTTC
TTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAAT
TCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGC
TGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCGTG
GACTACCAGGGGTATCTAATCCTGTTTGCTCCCCACGCTTTTCGAGCCTCAGCGTCA
GNTTACAGACCAAAGAGCCGCCTTCCCCCCTGGTGGTTCCTCCATATATCTACNCAT
TTCACCGCTACACATGGAATTCACTCTCCTCTTCTGCACTCANGTCTCCAGTTTCNAA
GAACCCTCCCCGGTNGAGCCGGGGGNTTTCNATCNNACTNAAAAACCNCNTGCCTN
CGCTTTACNCCCAANAATCCGGANACCNTGNCCCNTANTATNACCCNGGNTGCTGG
ACNTAATTACCCNGGTTTTTTGGTCAAANCCGNCAANGATNAAAGTNCNTTCCCCTT
91
NTTNTTTNTGAAAANNAATTTNCAANTCCAAAACCTTTTTACTCNCCGGGGTTGGNN
GGNAAAATTTNNCATTGCCAAAATCCCCTATTGGCCCCCNGAGAATTGGGCCGGTNC
AANCCA
Eub 2 27 FB
GANGACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCTTCTTTCCCACCCCAG
CTTGCTCCACCGGGAAAGAGGAGTGGCGGACGGGTGAGTAACACGTGGGCAACCTG
CCCTTCAGCGGGGGATAACACTTGGAAACAGGTGCTAATACCGCATAATACCTCAG
AGCACATGCTCAAAGGTTGAAAGACGCTTTCGGGTGTCACTGGAGGATGGGCCCGC
GGTGCATTAGCTAGTTGGTGAGGTAACGGCCCACCAAGGCCACGATGCATAGCCGA
CCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGA
GGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGT
GAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTCAGAGAAGAACAAGGGTGA
GAGCAACTGTTCACCCCTTGACGGTATCTGGCCAGAAAGCCACGGCTAACTACGTGC
CAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAA
GCGAGCGCAGGCGGTCCCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAG
GGTCATTGGAAACTGGGGGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGT
AGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGG
TCTGTAACTGACGCTGANGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCT
GGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTNCCCCCCTTCAG
TGCTGCAGCTAACGCATTAAGCACTCCCCNTGGGGAGTACCACCCCANGNTNAAAC
TCAANGAATTGACGGGGGCCCCCCAAGCGGTGGAACTGTGGTTTATTCNAACAACC
CGAAAACCTTACNGGTTTGAATCNTTNGACCCCCTANAATANGGNCTCCCCTTCGGG
GCAAAAAACNGGNGGGGATGNTTTCCNCACTCNGTCCGGAATNTTGGTTAATNCCC
NACCAGCCCACCTTNTNTTNGNTGCNTNTTNAATTGGNCCNTTNANATGCCGGGANA
ACCGGGGAGGGGGGTAACNTAN
Eub 2 1492 RS
CCCATTCATCTGTCCCACCTTAGGCGGCTGGCTCCATAAATGGTTGCCCCACCNACT
TNGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACG
TATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGCAGGCGA
GTTGCAGCCTGCAATCCGAACTGAGAGAGGTTTTAAGAGATCCGCTTGGCCTCGCGG
CTTCGCTTCTCGTTGTACCTCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGG
CATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCGCTAG
AGTGCCCAACTGAATGATGGCAACTAACGATAAGGGTTGCGCTCGTTGCGGGACTT
AACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCTCTTTGCC
CCCGAAGGGGAGGCCCTATCTCTAGGGCGGTCAAAGGATGTCAAGACCTGGTAAGG
TTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCA
ATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTA
GCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGGCGT
GGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAGCGTCAG
TTACAGACCAGANAGCCGCCTTCGCCACTGGTGTTCCTCCATATATCTACGCATTTC
ACCGCTACACATGGAATTCCACTCTCCTCTTCTGCACTCAAGTCCCCNAGTTTCCAAT
GACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAAACTTAAGGGACCGCCTGCCCT
CCCTTTACCCCAANAAATCCGAAAACGCTNGCNCCTACGATNACCCGGCTGCTGGA
CGNANTTAGCCNNGGTTTNTGGCCAANCCGTCAGGGGNAANNNTGTTNNCCCTTGT
92
NTTNTTGAANANNTTTTCANCCNAANCTTTTTACTCCNNGGGGTGTCGGGNAAATTT
NNCATGCNAAATCCTNTGTGCNCCGAAANTTGGGCCGGTTTANCCANGGG
Eub 3 27FB
NNNNNNNCGCTGTNCGANGGNCCCNACGNGCGCCATACGTAACAAGGTAACCGTCG
ACGGCCATTTTANACNAANNGNGGGACGGGTGAGTAACACGTGGGTAACCTACCCT
TTAGTGAGGGATAACACTTGGAAACAGGTGCTAATACCGCATAAAAGTCTAGTTCG
CATGAACAAGATTTGAAAGACGCTTTACGGTGTCACTAAAGGATGGACCCGCGGTG
CATTAGTTAGTTGGTGAGGTAACGGCTCACCAAGACGATGATGCATAGCCGACCTG
AGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCA
GCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGT
GAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGT
AACTGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGC
AGCCGTGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGA
GCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCTGGCTTAACCGGGGAGGGTCA
TTGGAAACTGGGGAACTTGAGAGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGG
TGAAATGCGTACATATATGGAGGAACACCACTGGCGAAGGCGGCTCTCTGGTCTGT
AACTGACGCTGATGTTCGAAAGCTAGGGGAGTAAAACATGATTAGATACCCTGGTA
GTCCACGTCGTAAACTATGAGTGCTAAGTTTGGAGGATATCCGTTCTTCAATGATGC
ATCTTAGCGCATTAAGCATCAGCCTGGGGAGAACGACCTAAATGTTATACTCAAAG
GATTGACGGGGGCCCGCCTAGGTTTAGAGAAGTGGTTTAATTCGATCCACGAGAAC
AACTTATCAGGCTTTGACTTCTTTGTCCCTCTAGAGATGANCTTTCCTTTCGGGACAA
GGGTAAGCTGTTCTGGATTCGCCGTCCGTCCCGTGACTTTGGTCAATTCTCGCCACCA
TGCTAATCTCTTATGTCGCTGCCATCCTTTTATCCGGCCCCCNNCTACCTGTCNTGCA
TACCGGTAGGAAGGTGGGATGACATTAACCTCCTGTCCCTTTAGTAATCTTGCTTAC
Eub 3 1492RS
NNNNANNGNCGNAGCCGACCACTCCTGCATCCTCAACCAGGATCAAACTCTGGATC
CCGCANGTCATCNTGAAAGGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTTTTC
ACCGCGGCGTGCTGATCCGTAATTACTAGCGATTCCGGCTTCATGTAGGCGAGTTGC
AGCCTACAATCCGAACTGAGAGAAGCTTTAAGAGATTAGCTTGATCTTGCGATTTCG
CGACTCGTTGTACTTCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGA
TGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCGCTAGAGTGC
CCAACTAAATGATGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGACTTAACCC
AACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTTTGTCCCCGA
AGGGAAAGCTCTATCTCTAGAGTGGCCAAAGGATGTCAAGACCTGGTAAGGTTCTTC
GCGTTGTTTCAAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCC
TTTGAGTTTCAACCTTGCGGCCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTG
CAACACTGAAGGGCGGAAACCCTCCAACACATACACTCATCGTTTACGGCGTGGAC
TACCAGGTTATCTAATCCTGTTTGCTTCCCACGCTTTCGAGTCTCAGCGTCAGTTACA
GACTAGAGAGCCGCCTTCGCCATTGGTGTTCCTCCATATATCTACGTCATTTCCCGTT
ACACATGGAATTTCCACTTTTCCTCTACTGCATTAAAGT
93
Eub 4 27FB
NNNNCTACGCTGGAGANGGTACCACACCTGCGACAACGTAACAAGGTAACCGTCAA
CGGCCATTAGGATCTTNTANGGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGG
GTGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGTGG
GGGACCTTCGGGCCTCATGCCATCAGATGTGCCCAGATGGGATTAGCTGGTAGGTGG
GGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCAC
ACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGC
ACAATGGGCGCAAGCCTGATGCATTCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTT
GTAAAGCACTTTTAGCGGGGAGGAATGCGTTGAGGTTAATAACCTCTCCGATTGACG
TTACCCGCAGAAGAAGCACCGGCTAATTCCGTGCCAGCAGCCGCGGTAATACGGTG
GGTGCAAGCGTTAATCGGAATAACTGGGCGTATAGCGCACGTAGGCGGACTGTTAA
GTCGTATGTGAAATCCCCGGGCTCAACCTGGGAACTGCTTTCTAAACTGGCNGGTTA
GAGTCTTGTTGAGGGTGTTGAATTCAGGATTAGCGGTAAAATGAAAGAGATCTGGA
AGATACCGGATTAAAGGCGTTCCCTGTACAAAGACTGACTCTCATTTGCCAAGCGTG
GGTAGCAAACTNTT
Eub 4 1492RS
GNNGNCNTNNNCGNNNGACTGNCTGCATCTNCAGCCAGGATCAAACTCTGGATCCC
GCANNNCGCGTNANGGGGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTTTAANC
CGAAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAG
ACTCCAATCCGGACTACGACATACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCT
TCTCTTTGTATATGCCATTGTAGCACGTGTGTAGCCCTGGTCGTAAGGGCCATGATG
ACTTGACGTCTTCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGATTTCCC
GGCCTAACCGCTGGCAACAAATGATAAGGGTTGCGCTCGTTGCGGGATTTAACCCA
ACATTTCACAACACGAGCTTACGACAGCCATGCAGCACCTGTCTTTCAGTTCCCGAA
GGCACCAATCCATCTCTGGAAAGTTCTGTGGATGTCAAGACCAGGAAAGGTTCTTCT
CGTTGCATCAATTTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCAT
TTTAGTTTTAACCTTGCTGCCGTTCTCCCCAGGTTGTTTTTTTAACGCGTAACTCCGG
AAGCCAN
Eub 5 27FB
NNNNNACGCTGGCGGCAGGCCTACACATGCAAGTCGGGCGGGAACAGGTCTTTCGG
GAAGCTGACGAGCGGCGGACGGGTGAGTAAAGTCTAGGAATCTGCCCAGTAGTGGG
GGACAACGCGGGGAAACTCGCGCTAATACCGCATACGCCCTACGGGGGAAAGGTGC
TTTATTGTACCGCTATTGGATGAGCCTAGATCAGATTAGCTAGTTGGTGGGGTAAAG
GCCTACCAAGGCGACGATCTGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGA
CTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGG
GGGCAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAG
CACTTTAAGTTGGGAGGAAGGGGTAGAGTAATGACCTACTTTTGACGTTACCAACAG
AATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGTGCAAGCG
TTAATCGGAATAACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTCAAGTTGGATGTGA
AATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGACTGACTAGAGTACGGTA
GAGGTTAGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACAC
CAGTGGCGAAGGCGACTAACTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGG
GAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTCAACTAGCC
94
GTTGGGACCCTTGAGGTTTTAGTGGCGCAGCTAACGCAATAAGTTGACCGCCTGGGG
AGTACGGTCGCAAGATTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTG
GAGCATGTGGTTTAATTCGACGCAACGCGAAGAACCTTACCTGGGCCTTGACATGTC
CGGAATCTTGCAGAGATGCGAGAGTGCCTTCGGGAATCGGAACACAGGTGCTGCAT
GGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGTAACGAGCGCAACC
CTTATCTTAGTTACCAGCACGTTATGGTGGGCACTTTANNGACTGCNGTGACAACGA
GANGTGGGNTGACGTCAGTCATCATNNCCTTACGNAGGCTACNNGTGCTACATNNN
GNACAGAGGGTCGNNNCTNNNNGANCTATCTCAGAAAACNATCNNNNCNNATTGA
NNNGNAACTCGNNCTTNATGAANNGGAATCGCNNNAATCCNNANCNAATNTNCNCG
GGNA
Eub 5 1492RS
NNNNNCAGTCTGATCCTCCGTGGTAACCGGCCTCACGAGGTTAGCCTAGCTACTTCT
GGAGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTAT
TCACCGCGACATTCTGATTCGCGATTACTAGCGATTCCGACTTCATGAAGTCGAGTT
GCAGACTTCAATCCGGACTACGATTGGTTTTCTGAGATTAGCTCCACTTTACAGTTTG
GCGACCCTCTGTACCAACCATTGTAGCACGTGTGTAGCCCTGGCCGTAAGGGCCATG
ATGACTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAAAGTG
CCCACCATAACGTGCTGGTAACTAAGGATAAGGGTTGCGCTCGTTACGGGACTTAAC
CCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGTGTTCCGATTCCC
GAAGGCACTCTCGCATCTCTGCAAGATTCCGGACATGTCAAGGCCAGGTAAGGTTCT
TCGCGTTGCGTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATT
CATTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCAACTTATTGCGTTAGCT
GCGCCACTAAAACCTCAAGGGTCCCAACGGCTAGTTGACATCGTTTACAGCGTGGA
CTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTCAGTGTCAGTATC
AGTCCAGTTAGTCGCCTTCGCCACTGGTGTTCCTTCCTATATCTACGCATTTCACCGC
TACACAGGAAATTCCACTAACCTCTACCGTACTCTAGTCAGTCAGTTTTGGATGCAG
TTCCCAGGTTGAGCCCGGGGATTTCACATCCAACTTGACAAACCACCTACGCGCGCT
TTACGCCCAGTTATTCCGATTAACGCTTGCACCCTTCGTATTACCGCGGCTGCTGGCA
CGAAGTTAGCCGGTGCTTATTCTGTTGGTAACGTCAAAGTAGGTCATTACTCTACCC
CTTCCTCCCAACTTAAAGTGCTTTACATCCGAAGACCTTCTTCACACACGCGGCATG
NTGGATCAGGGNGCCCCCATTGTCCAATATTCCCCACTGCTGCCTCCCGTAGAGTCT
GGNNNNGTCTCAGTCCCAGTGNNNNGATCATCCTNCTCAGAACAGCTACAGAATCG
TCCGCCTNNNNNNTTACCNNNCNACTAGCNTANNTGANTAGNNNNTCNNNCGGNNN
CANAAGNNCCTTCCCNNNAGNNGNANTGCGGNATNTNN
Eub 6 27FB
NNNNNNNACGCTGGCGGCGTGCTACACATGCAAGTCGAACCANTGTGTTTCTTGTCC
CGGNGCTTGCTCCACCGAGAGAAAAAGAGTGGCGAACGGGTGAGTAACACGTGGGT
AACCTGCCCACCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACA
ATAGAAGCCGCATGGTTTTTATTTGAAAGGCGCTTTTGCGTCACTGGTGGATGGACC
CGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGC
CGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACG
GGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCG
CGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGA
95
TGAGAATAAAATGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTAC
GTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGT
AAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGG
GAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATG
TGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTC
TGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATAC
CCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTC
AGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGA
AACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCG
AAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGA
GCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCG
TGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCAT
TTAGTTGGGCACTCTAGCGNNACTGCNGTGACAAACCGNNNNGTGNGATGACGTCA
AATCATCATNNNNTANNACCTGGNNTACNNNCNNGNNNCAATGGNNNNTACANCN
NNNNN
Eub 6 1492RS
NNNNNANGATCAACTATGCCCCTTAGGCGGCTCTGTCCCTAGAGCAAACTCTGGATC
CTGCGGGGGTTACAAACTTTCGGGGGGTGACGGGCGGGGTGTACAAGGCCCGGGAA
CGTATTCACCGCGGCGGGCTGATCCGCGATTACTAGCGATTCCGGCTTCATGTAGGC
GAGTTGCAGCCTACAATCCGAACTGAGAGAAGCTTTAAGAGATTAGCTTAGCCTCGC
GACTTCGCAACTCGTTGTACTTCCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGG
GGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCGCT
AGAGTGCCCAACTAAATGATGGCAACTAACAATAAGGGTTGCGCTCGTTGCGGGAC
TTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTTTG
TCCCCGAAGGGAAAGCTCTATCTCTAGAGGGGTCAAAGGATGTCAAGACCTGGTAA
GGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCG
TCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCG
TTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCACTCATCGTTTACGG
CGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAGCGT
CAGTTACAGACCAGAGAGCCGCCTTCGCCACTGGTGTTCCTCCATATATCTACGCAT
TTCACCGCTACACATGGAATTCCACTCTCCTCTTCTGCACTCAAGTTTCCCAGTTTCC
AATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGC
GCTCGCTTTACGCCCAATAAATCCGGACAACGCTTGCCACTTACGTATTACCGCGNC
TGCTGGCACGTAGTTAGCCGTGNTTTCTGGTTAGATACCGTCAAGGNTGAACATTTT
ACTCTCATCCTGTNTTCTCTACAACAGAGTTTACGATCGGAAACCTCTCANTCACGC
GNGTGCTCGNCAGACTTTCGTCATGGCGAGATCCTACTGNTGCCTNCGTAGANTNNN
NGNNNCANTCNNNNNNATTCNCCTCTNAGNCGNNNCATCNTGCTNNNACNTACTNC
ACTGNNATGNNNNGNNCCATNTCCNCNGNACGNNAAANNNTN
Eub 7 27FB
GNNNNNNNNGCTGNNCGNCNNGCCTANCACATGCGAGTCGTAACAAGGTAACCGTC
GACGGCCNCNTNTNCGTNNGGGN
96
Eub 7 1492RS
NNNNNNNNNNNAGNNNNNCNACTGTTGTGCGNCCTGAAGCCAGGATCAAACTCTGG
ATCCCGCA
Eub 8 27FB
NNNNNNNACGCTGAAGATGCTTACACATGCAAGTCTACTTGATCCTTCGGGTGAAG
GTGGCGGACGGGTGAGTAACGCGTAAAGAACTTGCCTTACAGACTGGGACAACATT
TGGAAACGAATGCTAATACCGGATATTATGATTGGGTCGCATGATCTGGTTATGAAA
GCTATATGCGCTGTGAGAGAGCTTTGCGTCCCATTAGTTAGTTGGTGAGGTAACGGC
TCACCAAGACGATGATGGGTAGCCGGCCTGAGAGGGTGAACGGCCACAAGGGGACT
GAGACACGGCCCTTACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGAC
CAAAAGTCTGATCCAGCAATTCTGTGTGCACGATGAAGTTTTTCGGAATGTAAAGTG
CTTTCAGTTGGGAAGAAGTCAGTGACGGTACCAACAGAAGAAGCGACGGCTAAATA
CGTGCCAGCAGCCGCGGTAATACGTATGTCGCAAGCGTTATCCGGATTTATTGGGCG
TAAAGCGCGTCTAGGCGGCTTAGTAAGTCTGATGTGAAAATGCGGGGCTCAACCCC
GTATTGCGTTGGAAACTGCTAAACTAGAGTACTGGAGAGGTAGGCGGAACTACAAG
TGTAGAGGTGAAATTCGTAGATATTTGTAGGAATGCCGATGGGGAAGCCAGCCTAC
TGGACAGATACTGACGCTAAAGCGCGAAAGCGTGGGTAGCAAACAGGATTAGATAC
CCTGGTAGTCCACGCCGTAAACGATGATTACTAGGTGTTGGGGGTCGAACCTCAGCG
CCCAAGCTAACGCGATAAGTAATCCGCCTGGGGAGTACGTACGCAAGTATGAAACT
CAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGACGC
AACGCGAGGAACCTTACCAGCGTTTGACATCCCAAGAAGTTAACAGAGATGTTTTCG
TGCCTCTTCGGAGGAACTTGGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCG
TGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTTTCGTATGTTACCATCAT
TAAGTTGGGGACTCATGCNAGACTGCNGCGATGAGCAGGAGAAGGTGGGGATGACG
TCAGTCATCATGCCCTTAATACGCTGGCTACCNCGTGCTACAATGGTAGTACAGAAN
NNCTGCAAACNGCCAGGTAGCTANNTCATAAAANTNTCTANNCGGANTGNNCCTNN
NNACTNGAGTANNNNNNTNNNTCGNCGTNNNNNATCNGCCAANNNNNNGCCTATTG
TTTNNN
Eub 8 1492RS
CNNNNCATCGCTATCACACCCTCGGAACATCCCTCCTTACGGTTAGGCCTGTTACTT
CAGGTGCAACCAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGACCCGAGAACGT
ATTCACCGCAACATAGCTGATTTGCGATTACTAGCGATTCCAACTTCATGTACTCGA
GTTGCAGAGTACAATCCGAACTAAGAATAGTTTTATGAGATTAGCTTACCCTCGCAG
GTTTGCAGCTCTCTGTACTACCCATTGTAGCACGTGTGTAGCCCAGCGTATAAGGGG
CATGATGACTTGACGTCATCCCCACCTTCCTCCTGCTCATCGCAGGCAGTCTCGCAT
GAGTCCCCAACTTAATGATGGTAACATACGAAAGGGGTTGCGCTCGTTGCGGGACTT
AACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTCACCAAGT
TCCTCCGAAGAGGCACGAAAACATCTCTGTTAACTTCTTGGGATGTCAAACGCTGGT
AAGGTTCCTCGCGTTGCGTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGTCCC
CGTCAATTCCTTTGAGTTTCATACTTGCGTACGTACTCCCCAGGCGGATTACTTATCG
CGTTAGCTTGGGCGCTGAGGTTCGACCCCCAACACCTAGTAATCATCGTTTACGGCG
TGGACTACCAGGGTATCTAATCCTGTTTGCTACCCACGCTTTCGCGCTTTAGCGTCAG
TATCTGTCCAGTAGGCTGGCTTCCCCATCGGCATTCCTACAAATATCTACGAATTTCA
CCTCTACACTTGTAGTTCCGCCTACCTCTCCAGTACTCTAGTTTAGCAGTTTCCAACG
97
CAATACGGGGTTGAGCCCCGCATTTTCACATCAGACTTACTAAGCCGCCTAGACGCG
CTTTACGCCCAATAAATCCGGATAACGCTTGCGACATACGTATTACCGCGGCTGCTG
GCACGTATTTAGCCGTCGCTTCTTCTGTTGGTACCGTCACTGACTTCTTCCCAACTGA
AAGCACTTTACATTCCGAAAACTTCATCGTGCACNNCAGAATTGCTGGATCAGACTT
TTGGNCATGTCANNTCCCNACTGCTGCCTCCGTAGAGTAGNNNTGTCTCAGTCCCNN
NNNNGTCACCNCTCAGCGNTACCATCATCGCTTNNNNNNGTACCTNNCANTACNAT
GNNNNNNNNNNNTCCAGGCAATATAGCNTTCTANANNNNNNNNNGNANCCAATN
Eub 9 27FB
NNNANGACGCTGGCGGCAGGCCTACACATGCAAGTCGAGCGGTAGCACAGAGAGC
TTGCTCTCGGGTGACGAGCGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGAT
GGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAA
AGTGGGGGACCTTCGGGCCTCATGCCATCAGATGTGCCCAGATGGGATTAGCTGGTA
GGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCA
GCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAAT
ATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTT
CGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGTGAGGTTAATAACCTTATCG
ATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAA
TACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGT
CTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGG
CAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTA
GAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTC
AGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTA
AACGATGTCGATTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTT
AAATCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGG
GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTA
CCTGGTCTTGACATCCACAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTGT
GAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTGTGAATGTGGGTAGTCCCGCAC
GAGCGCACCCTNTCTTTGTGCCAGGCGGTCAGGCCGGGNCTCAAGGAGACTGCAGT
GATAACTGAGAGGTGGGGATGACGTCCAGTCATCATNNNNCGACAGGGCTANCACN
TGCTACATGCNTTNNNANGAGAANNNACTNNNNNNAGCGNCNNANNNNGTCGTATC
GATGGNNNNNTNNANNGANNTCNANNNNANNNNNGN
Eub 9 1492RS
NNNNNAGNTATGATNCAAGTGGTAAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCT
TTTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATT
CACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTG
CAGACTCCAATCCGGACTACGACATACTTTATGAGGTCCGCTTGCTCTCGCGAGGTC
GCTTCTCTTTGTATATGCCATTGTAGCACGTGTGTAGCCCTGGTCGTAAGGGCCATG
ATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTT
CCCGGCCTAACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAAC
CCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCACAGTTCCC
GAAGGCACCAATCCATCTCTGGAAAGTTCTGTGGATGTCAAGACCAGGTAAGGTTCT
TCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATT
CATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGATTTAACGCGTTAGCT
98
CCGGAAGCCACGCCTCAAGGGCACAACCTCCAAATCGACATCGTTTACAGCGTGGA
CTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTT
TGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGATCTCTACGCATTTCACCG
CTACACCTGGAATTCTACCCCCCTCTACAAGACTCTAGCCTGCCAGTTTCGAATGCA
GTTCCCAGGTTGAGCCCGGGGATTTCACATCCGACTTGACAGACCGCCTGCGTGCGC
TTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGG
CACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATCGATGAGGTTATTAAAC
CTCACCGCCTTCCTCCCCGCTGAAAGTGCTTTACANCCGAAGGCTTCTTCANACACG
CGGCATGCTGCATCAGGCTGCGCCATGTGCAATATTCCCCACTGCTGCCTCCCGTAG
NANNNTGACNNNNCTCAGTTTCAGGTGGNGGNTNNNATCCTCTCAGAACAGNCTAG
GNTCNNCNCNAGNNNNNNNNNANCCNNNCNACNAGNCNTATCNNTCTGNNNAANT
TCTGNAANTGNNNTGANNGCCCNNNN
Eub 10 1492RS
NNNNNNNNNTNTAGNANCTCAACTGTTGAGCCCCCAACCGAGGGCTAAGCTTCCTA
CTCCTATTGCAACCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAAC
GTATTCACCGTAGCATTCTGATCTACGATTACTAGCGATTCCGACTTCATGGAGTCG
AGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCG
AGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTACTCGTAAGGG
CCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCTTT
GAGTTCCCGCCATTACGCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGA
CTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCTCA
GTTCCCGAAGGCACAAGACTGTCTCCAGTCTCTTCTGAGGATGTCAAGAGTAGGTAA
GGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCG
TCAATTCATTTGAGTTTTAATCTTGCGACCGTACTCCCCAGGCGGTCAACTTAACGCG
TTAGCTCCGGAGCCCACAGCTCAAGGCCGCAAACTCCAAGTTGACATCGTTTACAGC
GTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTC
AGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCACATCTCTACGCATTT
CACCGCTACACGTGGAATTCTACCCCCCTCTACGAGACTCTAGCTAACCAGTTTTTG
AATGCAGTTCCCAGGTTAAGCCCGGGGATTTCACATCCAACTTAAATTAACCGCCCT
GCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCCTCCGTATTACCGC
GGCTGCTGGCACGGAGTTAGCCCGGTGCTTCTTTTTGTCGGGTAACGGTCAATTGAA
TAAAGTATTTAATTTATCCTCCTTCCTTCCGACTGAAAAGGTGCTTTACAACCCTTAA
GGGCTTTCTTAACAAACGCCGGAATGGNTGCATCAGGNNNTCCCCATTGGGCATAAT
CCCCNCTNNCTGCCTNNCGGAAGNGTCTGGAANCGTGTCNTCAGTTCCAGGGTTGGN
NGNNNTTCNNNCCNAAACNNCNNNAGAATNNCNCNTAGGNNNNCCNTTNCCCCCAC
CTACTNNTTANNNCCNNNNNGGNTTCATTCTCTNNNNGNGNNNNN
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APPENDIX D: Phylogenetic Trees from BLAST
Eubacteria Sample #2 results of BLAST phylogenetic tree analysis
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Eubacteria Sample #3 results of BLAST phylogenetic tree analysis
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Eubacteria Sample #6 Results of BLAST Phylogenetic Tree Analysis
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Eubacteria Sample #9 results of BLAST phylogenetic tree analysis
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Eukarya Sample #2 results of BLAST phylogenetic tree analysis
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Eukarya Sample #3 results of BLAST phylogenetic tree analysis
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VITA
AMY RENÉE ROBERTSON
Personal Data:
Date of Birth: August 27, 1981
Place of Birth: San Bernardino, California
Marital Status: Single
Education:
Sullivan South High School, Kingsport, Tennessee, 1999
East Tennessee State University, Johnson City, Tennessee; Biology, B.S.,
2004
East Tennessee State University, Johnson City, Tennessee; Biology, M.S.,
2006
Professional
Experience:
Publications:
Graduate Assistant, East Tennessee State University, College of Arts and
Sciences, 2004-2006
None
Honors and Awards: Search Committee for New Department Chair, 2005-2006 Academic Year
The Chancellor’s List, 2006
William Harvey Fraley and Nina M. Fraley Research Award, 2006
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